Cyclophilin A regulates the apoptosis of A549 cells by stabilizing Twist1 protein

Epithelial cell migration, permeability and apoptosis play significant roles in the pathogenesis of several inflammation-related diseases and tumorigenesis (Fathimath Muneesa et al., 2021; Hayashi et al., 2006; Shen et al., 2016). Epithelial-mesenchymal transition (EMT), a complex reprogramming process of epithelial cells, plays a vital role in tumor invasion and metastasis (Chen et al., 2017; Ling et al., 2021). EMT is defined as a morphologic alteration from an epithelial to a mesenchymal phenotype, including a loss of epithelial cell markers, such as E-cadherin, α-catenin, and γ-catenin, and a gain in mesenchymal components, such as vimentin, N-cadherin and fibronectin (Huber et al., 2005; Wang et al., 2017; Yu et al., 2014). In addition, studies show that various transcriptional factors, such as Twist, Slug and Snail, regulate EMT (Yu et al., 2010; Zheng and Kang, 2014).

Twist1 is a basic helix-loop-helix transcription factor involved in a variety of normal biological processes, including embryogenesis, cellular motility and tissue differentiation (Bialek et al., 2004; Zhu et al., 2016). Mutations in Twist1 can be embryonically lethal or result in severe craniofacial defects associated with Saethre–Chotzen syndrome (Chen et al., 2020b; Finlay et al., 2015). Twist1 has also been reported to be a crucial contributor to cancer progression by enhancing EMT, promoting cancer stem cell phenotypes and causing drug resistance (Lu and Kang, 2019; Ren et al., 2016).

Phosphorylation and ubiquitylation are the most important post-translational modifications that affect Twist1 function by regulating the stability of Twist1 protein. Mitogen-activated protein kinases (MAPKs) phosphorylate Twist1 at Ser68 to block ubiquitin-mediated degradation of the protein (Hong et al., 2011). Activation of protein kinase B (AKT1) can mediate the phosphorylation of Twist1 on Ser42, leading to ubiquitylation-mediated degradation (Li et al., 2016). Casein kinase 2 (CK2) phosphorylates Twist at S18 and S20, which increases Twist1 stability in response to interleukin (IL)-6 stimulation (Su et al., 2011). It was also reported that phospholipase C (PLC)ε increased phosphorylation of Twist1 at Ser68 and stabilized the Twist1 protein through MAPK signaling (Fan et al., 2019). Pre-mRNA processing factor 19 (Prp19, also known as PRPF19) promoted the activation of p38 MAPK (also known as MAPK14), preventing Twist1 from degradation (Yin et al., 2016). Notably, regulation of the phosphorylation of Twist1 is an important way to control its stability.

Cyclophilin A (CypA, also known as PPIA), a key member of the immunophilin family, acts as the intracellular target of the immunosuppressive drug cyclosporin A (CsA) (Handschumacher et al., 1984a; Wang and Heitman, 2005) and a peptidyl-prolyl cis/trans isomerase (PPIase), and catalyzes the cis-trans isomerization of proline amidic peptide bonds, through which it regulates multiple biologic processes, such as intracellular signaling, transcription, inflammation and apoptosis (Brazin et al., 2002; Colgan et al., 2004; Lu et al., 2007; Ma et al., 2021). Our previous study showed that CypA could facilitate IFN responses through promoting K63-linked ubiquitylation of RIG-I and inhibiting K48-linked ubiquitylation of MAVS (Liu et al., 2017). Yet several lines of evidence indicate that extracellular CypA stimulates pro-inflammatory signals and MAPK, including ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), JNK (also known as MAPK8) and p38 (Jin et al., 2004; Suzuki et al., 2006). Therefore, determining the regulation of CypA on the ubiquitylation and phosphorylation of Twist1 is crucial for preventing pro-inflammatory diseases and tumorigenesis.

In this study, we hypothesized that CypA regulated the phosphorylation and transcriptional activity of the transcription factor Twist1, and the expression of its downstream genes. We found that CypA promoted Twist1 phosphorylation at Ser68 and transcriptional activity, and inhibited apoptosis in A549 cells. Moreover, CypA stabilizes Twist1 by suppressing the K48-linked ubiquitylation of Twist1 and enhances interaction between Twist1 and p65 (also known as RELA), as well as nuclear accumulation of Twist1, which represents a new facet of CypA in the host physiological process.

To investigate the impact of human CypA on Twist1 phosphorylation in A549 cells, we monitored Twist1 phosphorylation in short hairpin (sh)RNA-based CypA-knockdown A549 cells (A549/CypA⁻) and wild-type (WT) A549 cells (A549/CypA⁺), and found that CypA gene knockdown dramatically decreased Twist1 phosphorylation at Ser68 in a dose-dependent manner (Fig. 1A). We further determined the effect of CypA on Twist1 phosphorylation in CypA-overexpressed A549 cells. Our results demonstrated that CypA gene overexpression significantly increased Twist1 phosphorylation at Ser68 in a dose-dependent manner (Fig. 1B), indicating that CypA promotes Twist1 phosphorylation at Ser68 in A549 cells. We also used the CypA immunosuppressive inhibitor CsA to inhibit CypA activity. To this end, A549 cells were treated with 10 µM CsA for different time periods and with varying concentrations of CsA for 60 min. As shown in Fig. 1C,D, CsA treatment suppressed Twist1 phosphorylation at Ser68 in a time- and concentration-dependent manner by blocking CypA activity. To ensure the specificity of CypA inhibitor regulation of Twist1 phosphorylation, we used another non-immunosuppressive inhibitor of CypA, DEB025 (Alisporivir), in different concentrations. Consistent with the results of CsA treatment, DEB025 decreased Twist1 phosphorylation (Fig. 1E). The results of normalized Twist1 phosphorylation are shown in the corresponding quantification bar graphs, and data are from three biological replicates of one batch experiment. These data collectively suggest that CypA promotes Twist1 phosphorylation at Ser68 in A549 cells.

To further investigate whether intracellular CypA promoted Twist1 phosphorylation by associating with it in A549 cells, we performed co-immunoprecipitation analysis. Twist1 plasmid with Flag-tag and CypA plasmid with Myc-tag were transfected into 293T cells. The co-immunoprecipitation results showed that Twist1 interacted with CypA (Fig. 2A). We further observed the endogenous CypA-Twist1 interaction by performing co-immunoprecipitation with the anti-CypA or anti-Twist1 antibody in A549 cells (Fig. 2B,C). Next, to determine whether this association is mediated by the CypA active site, we performed co-immunoprecipitation in the presence of CsA compared with the DMSO control group. As shown in Fig. 2D, CsA treatment significantly attenuated the association between CypA and Twist1, indicating that CypA interacts with Twist1, partly depending on its PPIase active site. Moreover, we observed that the mutant CypA-R55A, which is 100-fold less active as a PPIase (Zydowsky et al., 1992), attenuated the CypA-Twist1 interaction (Fig. 2E). These data taken together confirm that CypA interacts with Twist1.

Nuclear and cytoplasmic translocation of Twist1 is a vital translational modification (Devanand et al., 2019). To determine whether CypA regulated the subcellular localization of Twist1, we carried out immunofluorescence analysis by using A549 cells. We observed that Twist1 was mainly located in the nucleus of A549/CypA⁺ cells (Fig. 3A) and the DMSO treatment group (Fig. 3C). However, nucleus localization of Twist1 was blocked in A549/CypA⁻ cells (Fig. 3A) or the CsA treatment group (Fig. 3C), indicating that CypA is a crucial regulator of the subcellular localization of Twist1. The percentage of A549 cells showing the nuclear and cytoplasmic localization of Twist1 per total number of cells was determined and displayed in quantification bar graphs (Fig. 3B,D).

Being a host transcription factor, Twist1 governs the transcription of associated genes after entering the nucleus via promoter activation (Meng et al., 2018). To determine whether CypA regulated Twist1 transcriptional activity associated with its subcellular localization, we conducted a luciferase assay. As shown in Fig. 3E,F, Twist1 reporter luciferase activity was significantly inhibited in A549/CypA⁻ (Fig. 3E) or A549/CypA⁺ cells pretreated with the CypA inhibitor CsA (10 µM) (Fig. 3F). In A549/CypA⁻ cells, the reconstitution of CypA obviously increased Twist1 reporter luciferase activity in a dose-dependent manner, indicating that CypA is a crucial regulator of Twist1 transcriptional activity.

Considering CypA-promoted Twist1 nuclear accumulation, and transcriptional activity, we examined the effect of CypA on Twist1 downstream genes, CDH1 and CDH2. We performed qPCR in A549/CypA⁺ and A549/CypA⁻ cells transfected with Twist1 small interfering (si)RNA (siTwist1) or negative control siRNA (siNeg). The results of the qPCR showed a robust increase in the expression of CDH1 and a significant decrease in the expression of CDH2 in A549/CypA⁻ compared with control cells (Fig. 4A,B). However, this difference in the expression of CDH1 and CDH2 genes was not significant in cells transfected with siTwist1 (Fig. 4A,B) or in cells expressing loss-of-function Twist1 (Twist1ΔDBD), but was significant in cells expressing wild-type Twist1 (Fig. 4C,D). Consistently, CsA significantly upregulated CDH1 and downregulated CDH2 in A549/CypA⁺ cells compared with control cells (Fig. 4E,F). However, this difference in the expression of CDH1 and CDH2 genes was not observed in cells transfected with siTwist1 (Fig. 4E,F). These results collectively indicate that CypA-mediated regulation of CDH1 and CDH2 transcription partly requires Twist1 transcriptional activity.

On the basis of the observation that CypA downregulated CDH1 and upregulated CDH2, which play a key role in tumor metastasis and viability (Mrozik et al., 2018; Song et al., 2019; Wang et al., 2018), we assessed the ability of the CypA-Twist1 signaling pathway to regulate A549 cell death by examining the viability of A549 cells in the presence of the protein synthesis inhibitor cycloheximide (CHX). No significant decrease was observed in the viability of A549/CypA⁺ and A549/CypA⁻ cells in the absence of CHX. However, CHX treatment significantly decreased the viability in A549/CypA⁻ cells compared with A549/CypA⁺ cells. Intriguingly, the effect of CypA on cell viability increased in Twist1-overexpressed A549/CypA⁻ cells (Fig. 5A). To examine whether A549 cell death was caused by apoptosis, we analyzed cell nuclei by performing fluorescence microscopy. No significant apoptosis induction was detected in the absence of CHX. However, as shown in Fig. 5B, CHX treatment significantly induced apoptosis in A549/CypA⁻, which exhibited brighter nuclear shrinkage that was associated with abnormal DNA chromatin condensation and nuclear fragmentation (indicated by arrows). Moreover, the overexpression of Twist1 diminished the abnormal condensation of DNA of A549/CypA⁺ cells, indicating that the CypA-Twist1 signaling pathway regulated A549 cell viability and apoptosis.

Caspases, a family of cysteine proteases, are capable of cleaving essential cellular substrates after aspartic residues, and are critical for the initiation and execution phases of apoptosis (Green and Kroemer, 1998). We next identified the effect of CypA on the expression levels of the apoptosis-related gene Caspase-3 in A549/CypA⁺ and A549/CypA⁻ cells transfected with siTwist1. As shown in Fig. 5C,D, significantly elevated Caspase-3 levels were observed in A549/CypA⁻, indicating that the CypA-Twist1 signaling pathway regulated the viability and apoptosis of A549 cells.

Notably, more Twist1 was observed in A549 cells than in cells transfected with siRNA targeting CypA (siCypA) when we assessed the protein expression levels in lysates (Fig. 1A). Therefore, we sought to examine whether CypA affects the stability of Twist1. We performed a CHX chase assay of transfected Twist1 in A549/CypA⁺ and A549/CypA⁻ cells and found that CypA indeed inhibited the degradation of exogenous and endogenous Twist1 (Fig. S1A,B). In addition, the reconstitution of CypA promoted the stability of Twist1 in A549/CypA⁻ cells (Fig. S1C). Proteasome-, lysosome- and autophagosome-dependent pathways are principally responsible for intracellular protein degradation (Chen et al., 2020a), so specific protein inhibitors were applied to examine pathways involved in CypA-mediated Twist1 stabilization. In A549/CypA⁺ and A549/CypA⁻ cells, the proteasome inhibitor MG132 significantly inhibited the degradation of Twist1, whereas the lysosome inhibitor NH₄Cl and autophagosome inhibitor 3-methyladenine (3MA) did not (Fig. 6A), indicating that the degradation of Twist1 is controlled by ubiquitin-meditated proteolysis. Collectively, these data showed that CypA plays a critical role in suppressing proteasome degradation of Twist1.

Because Twist1 degradation in A549 cells was blocked by the proteasome inhibitor MG132, we further investigated the effects of CypA on Twist1 ubiquitylation. The ubiquitylation assay indicated that overexpression of CypA strikingly reduced the ubiquitylation of Twist1 (Fig. 6B). Consistently, in co-immunoprecipitation experiments, we also found that CypA decreased the ubiquitylation of Twist1 with or without MG132 stimulation (Fig. 6C; Fig. S1D). We next evaluated the types of ubiquitin chains of Twist1 that are mediated by CypA. Using K48-linkage specific polyubiquitin antibody and ubiquitin mutants that contain only one lysine residue, we found that CypA could attenuate the K48-linked but not K63-linked ubiquitylation of both exogenous and endogenous Twist1 (Fig. 6D; Fig. S1E). Taken together, CypA stabilizes Twist1 by suppressing the K48-linked ubiquitylation of Twist1.

Phosphorylation at Ser68 of Twist1 by MAPK has been reported to increase Twist1 stability in breast cancer cells (Hong et al., 2011). To determine whether Ser68 was the key site for CypA-mediated Twist1 stability, we mutated Ser68 to alanine (S68A). More importantly, any appreciable differences in the stability and ubiquitylation of Twist1-S68A mutant was not observed in the presence or absence of CypA (Fig. 6E,F), clearly indicating the essential role of CypA in the regulation of Twist1 stability. Kinases p38, c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinases1/2 (ERK1/2) have been shown to mediate the phosphorylation of Twist1 at Ser68 (Hong et al., 2011), so it is important to identify the specific kinase involved in CypA-mediated Twist1 phosphorylation. Then, we further blocked ERK, JNK and p38 using their respective specific kinase inhibitors, PD98059, SP60125 and SB203580. The western blotting results showed that all three inhibitors effectively antagonized their corresponding targets, and the absence of CypA impaired the stability of Twist1 in the presence of SB203580 but not with PD98059 and SP60125 (Fig. 6G), which suggested that CypA regulates Twist1 proteasomal degradation via p38. Consistently, p38 obviously enhanced the stability and phosphorylation of Twist1 in the presence of CypA (Fig. 6H). Collectively, these results suggest that CypA promotes the stability of Twist1 via the p38 MAPK/Twist1 pathway.

In our previous studies, we have demonstrated that CypA is critical for SeV-induced activation of NF-κB signaling pathways (Liu et al., 2017). The NF-κB signaling pathway triggered by tumor necrosis factor (TNF)-α and IL-1β has been intensively studied. To further investigate the impact of CypA on TNF-α- and IL-1β-induced NF-κB activation, a reporter assay was performed in 293 T/CypA⁻ and 293 T/CypA⁺ cells. We found that NF-κB-responsive luciferase activity induced by TNF-α or IL-1β stimulation was significantly lower in the absence of CypA (Fig. S2A,B). Similar results were obtained in A549 cells (Fig. S2C,D). Conforming to this observation, qPCR experiments showed that TNF-α- or IL-1β-induced transcription of NF-κB downstream genes, such as TNFA, IL-6, CXCL8 and CXCL1, was markedly reduced in A549/CypA⁻ and 293T/CypA⁻ cells (Fig. S2E,F). In addition, the phosphorylation of IKK α/β and IκBα, as well as the degradation of IκBα (Fig. S2G,H), was distinctly attenuated in TNF-α- or IL-1β-triggered A549/CypA⁻ cells compared with A549/CypA⁺ cells. Collectively, our results suggest that CypA upregulates TNF-α- and IL-1β-triggered NF-κB activation pathways.

It has been reported that p65 increased the nuclear expression of Twist1 and interacted with Twist1 after TNF-α stimulation (Roberts et al., 2017; Yu et al., 2014). In addition, previous studies have pointed out that CypA interacts with p65 (Sun et al., 2014). Thus, we wanted to explore whether CypA regulates the Twist1-dependent expression of CDH1 and CDH2 genes through the NF-κB signaling pathway. Our data showed that A549/CypA⁻ cells transfected with siTwist1 and sip65 significantly upregulated CDH1 and downregulated CDH2 compared with A549/CypA⁺ cells (Fig. 7A,B). The nuclear accumulation of endogenous Twist1 in A549/CypA⁺ and A549/CypA⁻ cells transfected with sip65 was detected using confocal microscopy. The results confirmed that the nucleus localization of Twist1 was blocked in A549/CypA⁻ cells transfected with sip65 (Fig. 7C), indicating that CypA regulated the subcellular localization of Twist1 through p65.

It has been reported that Twist1 interacts with p65 to regulate TNF-α-mediated EMT (Li et al., 2012; Roberts et al., 2017; Yu et al., 2014). We performed co-immunoprecipitation assays to explore the effect of CypA on the interaction between Twist1 and p65. Overexpression and co-immunoprecipitation assays showed that CypA promoted Twist1-p65 interaction (Fig. 7D). Accordingly, we observed similar results of endogenous interaction between Twist1 and p65 upon TNF-α stimulation (Fig. 7E). Confocal microscopy experiments indicated that CypA facilitated the recruitment of Twist1 and p65 to the nucleus upon TNF-α stimulation (Fig. 7F). These results taken together suggest that CypA increases Twist1-p65 complex formation and nuclear accumulation, which downregulated CDH1 and upregulated CDH2 expression.

CypA acts as the primary intracellular target of the immunosuppressive drug CsA (Handschumacher et al., 1984) and a key modulator of certain biological processes (Lu et al., 2007). However, the roles of CypA in the CypA-Twist1 signaling pathway are not well understood. In the present study, we found that CypA promoted Twist1 phosphorylation at Ser68, transcriptional activity and inhibited CHX-induced apoptosis in A549 cells. Furthermore, CypA interacted with Twist1 and inhibited the K48-linked ubiquitylation of Twist1. In addition, CypA increased the interaction between Twist1 and p65, which facilitated Twist1 nuclear accumulation and regulated Twist1-dependent expression of CDH1 and CDH2.

EMT, a complex reprogramming process of epithelial cells, plays an important role in tumor invasion and metastasis (Scott et al., 2019). Currently, studies show that EMT is controlled by various transcriptional repressors, such as Zeb-1/2, Slug, Snail and Twist1 (Hussen et al., 2021). Twist1, known as a master regulator of morphogenesis, induces EMT to facilitate breast tumor metastasis (Kwok et al., 2005). In addition to this in patients with breast carcinoma, high expression of Twist1 also correlates with tumor invasion and metastasis in patients with esophageal squamous cell carcinomas (Yuen et al., 2007), hepatocellular carcinoma (Matsuo et al., 2009), gliomas (Elias et al., 2005) and lung carcinoma (Jin et al., 2012; Liu et al., 2020). In this study, we found that CypA facilitated Twist1 phosphorylation at Ser68 and transcriptional activity. As one of the EMT-inducing transcription factors, Twist1 enhances the transcription of EMT-associated genes via promoter activation or repression, downregulating the epithelial phenotype-related genes, such as E-cadherin (CDH1), and upregulating the mesenchymal cell phenotype-related genes, such as N-cadherin (CDH2) (Shibue and Weinberg, 2017). Several lines of evidence indicate that N-cadherin can attenuate apoptosis by which N-cadherin-mediated cell contact maintains calcium homeostasis (Liu et al., 2021; Peluso, 1997). E-cadherin engagement can augment apoptosis stimulation through which E-cadherin interacts with death receptors DR4 and DR5 to promote the assembly of the death-inducing signaling complex (Geng et al., 2012; Lu et al., 2014). Here, we found that CypA induced Twist1-dependent expression of CDH2 and suppressed CDH1 expression, suggesting a positive role for CypA in Twist-mediated gene expression.

Twist1 promotes cell proliferation and migration, while inhibiting cell apoptosis and differentiation at appropriate developmental stages (Yang and Weinberg, 2008). In the present study, we observed that the overexpression of Twist1 diminished the CHX-induced apoptosis in A549/CypA⁺ cells, suggesting a protective role for CypA in Twist1-mediated apoptosis in A549 cells. The cleaved form of Caspase-3 is a well-known marker of apoptosis (Jacobsen et al., 1996). Previous findings also indicated that Twist1 knockdown stimulated Caspase-3 activation and apoptosis (Jen et al., 2020). In the present study, elevated levels of the cleaved form of Caspase-3 were observed in A549/CypA⁻ cells transfected with siTwist1. All these data indicate a protective role for CypA in the Twist1-mediated apoptosis of A549 cells.

Ubiquitylation affects many cellular processes, signaling pathways and disease states (Zhao et al., 2020). Twist1 can undergo post-translational ubiquitylation and consequent degradation under the influence of the ubiquitin-proteasome system, thereby controlling the intracellular levels of Twist1 (Kang et al., 2021). A series of studies have shown that some host proteins, such as HR23A and Pirh2, participate in the K48-linked ubiquitylation and degradation of Twist1 (Yang-Hartwich et al., 2019; Yu et al., 2019). In this study, we found that CypA inhibited the K48-linked ubiquitylation of Twist1 to slow down the degradation of Twist1, thereby facilitating Twist1 stability. Aside from ubiquitylation, phosphorylation is also the most important post-translational modification, which affects Twist1 function by regulating the stability of Twist1 protein. Phosphorylation at Ser68 of Twist1 by MAPK has been reported to increase Twist1 stability in breast cancer cells. The Twist1 S68A mutant results in increased ubiquitylation and subsequent degradation of Twist1, which controls EMT, and invasion in breast cancer cells depends on Twist1 stability (Hong et al., 2011). Here, we found that CypA could influence the ubiquitylation and stability of Twist1-WT but not Twist1-S68A, indicating that Ser68 was the key site for CypA-mediated Twist1 stability. Several studies also reported that CypA is associated with the activation of a variety of cancer-related signaling pathways, including the MAPK pathway (Kim et al., 2015; Li et al., 2018; Xiao et al., 2016; Yang et al., 2008). Our results indicated that CypA impaired the stability of Twist1 in the presence of SB203580, a specific inhibitor of p38-MAPK, thereby facilitating p38-mediated Twist1 stability. All these data suggest that CypA could mediate the phosphorylation of Twist1 at Ser68 via p38 MAPK, which inhibits its ubiquitylation-mediated degradation.

It has been well established that NF-κB signaling acts as a critical inflammatory mediator in response to invading pathogens. A large number of studies have shown that Twist1 interacts with p65, and the expression of the human Twist1 gene is directly upregulated by p65-mediated transcriptional activation in response to chronic inflammation (Li et al., 2012; Roberts et al., 2017; Yu et al., 2014). A previous study has pointed out that CypA interacts with the NF-κB subunit p65 and contributes to NF-κB activation signaling upon virus infection (Sun et al., 2014). Our results confirmed that CypA facilitates TNF-α- and IL-1β-induced NF-κB activation, and enhances Twist1-p65 interaction and nuclear accumulation, upregulating the expression of CDH2 and downregulating the expression of CDH1.

To conclude, our data demonstrate that CypA promotes Twist1 phosphorylation at Ser68, nuclear accumulation and transcriptional activity. On the one hand, CypA stabilizes Twist1 by suppressing its ubiquitin-mediated proteasome degradation, but on the other hand, CypA increases the Twist1-p65 interaction and Twist1 nuclear accumulation. Finally, CypA inhibits Twist1-mediated apoptosis of A549 cells. Hence, our data further expand the biological functions of Twist1 post-translational modification and downstream target gene transcription, which are essential for tumor metastasis and the function and regulatory role of CypA in this process. An understanding of the functions of CypA and Twist1 through MAPK p38-mediated ubiquitylation and the NF-κB signaling pathway may help the development of clinical therapies targeting CypA and Twist1 for treating cancer.

TRIzol (15596-026, Invitrogen, Carlsbad, CA, USA), TB Green (R820A, Takara, Tokyo, Japan), recombinant human TNF-α (DC008, Novoprotein, Shanghai, China), recombinant human IL-1β (200-01B, PeproTech, Rocky Hill, NJ, USA) and DEB025 (HY-12559, MedchemExpress, NJ, USA) were purchased from the indicated manufacturers. SP600125 (1496), PD98059 (1213), and SB203580 (1202) were obtained from Tocris (Ellisville, MO, USA); M-MLV (M1701) and a luciferase assay kit (E1980) were purchased from Promega (Madison, WI, USA); and MG132 (M7449), CHX (66819), DAPI (D9542) and 3MA (M9281) were purchased from Sigma-Aldrich (St Louis, MO, USA).

For immunoblot analysis, the following antibodies were used: rabbit polyclonal antibodies specific for human CypA generated as described previously (1:2000) (Liu et al., 2012); anti-c-Myc (1:2000, C3956) and anti-Flag M2 (1:2000, F3165, Sigma-Aldrich); anti-β-Actin (1:2000, 4970S), anti-HA (1:2000, 2999), anti-p65 (1:1000, 6956), anti-Caspase-3 (1:1000, 9664), anti-p65 (1:1000, 8242) and anti-K48-linkage-specific polyubiquitin (1:1000, 4289) were obtained from Cell Signaling Technology (Danvers, MA, USA); anti-Twist1 (1:1000, PA5-116628) and anti-p-Twist1 (1:1000, PA5-105703) were obtained from Thermo Fisher Scientific (Waltham, MA, USA); and anti-GAPDH (1:2000, c-25778), anti-β-Tubulin (1:2000, sc-5274), anti-mouse IgG (1:5000, sc-137075), and anti-rabbit IgG (1:5000, sc-2357) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

shRNA-based knockdown of CypA in human embryonic kidney 293T (293T/CypA⁻) cells and human lung carcinoma A549 (A549/CypA⁻) have been described previously (Liu et al., 2012). Cells were maintained in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO). All experiments were performed with mycoplasma-free cells.

Flag-Twist1 were constructed by standard molecular biology techniques. Flag-CypA, Myc-CypA, Flag-p65, HA-Ub and HA-K48-Ub have been described previously (Liu et al., 2017).

Twist1, p65 and negative siRNAs (siTwist1, sip65 and siNeg, respectively) were synthesized by GenePharma (Shanghai, China). The target sequences of these siRNAs are as follows: Twist1 sense, 5′-GGACAAGCUGAGCAAGAUU-3′, and Twist1 antisense, 5′-AAUCUUGCUCAGCUUGUCCUU-3′; p65 sense, 5′-AAACUCAUCAUAGUUGAUGGUGCUC-3′, and p65 antisense, 5′-GAGCACCAUCAACUAUGAUGAGUUU-3′; CypA sense, 5′-GCUCGCAGUAUCCUAGAAUTT-3′, and CypA antisense, 5′-AUUCUAGGAUACUGCGAGCTT-3′; and negative sense, 5′-UUCUCCGAACGUGUCACGU-3′, and negative antisense, 5′-ACGUGACACGUUCGGAGAA-3′. All cells in this study were transfected with the siRNAs using Lipofectamine RNAiMAX (13778075, Invitrogen) and with the respective plasmids using Lipofectamine LTX reagent with PLUS reagent (15338030, Invitrogen), according to the manufacturer's instructions.

Cells (1.5×10⁵) from different cell lines were transfected in a 24-well plate with Twist1 firefly luciferase and β-Gal plasmid together with other plasmids containing genes of interest using Lipofectamine 2000 (11668019, Invitrogen). Twenty-four hours later, cells were lysed in lysis buffer. After centrifugation at 12,000 g for 15 min at 4°C, the supernatants were stored at −80°C. Luciferase assays were performed using a luciferase assay kit (Promega).

Cell viability was measured with a CCK-8 assay. Briefly, A549 cells were seeded in 96-well plates and transfected with Flag-Twist1, and treated with CHX for 24 h at 37°C. After treatment, 10 μl CCK-8 solution (C0037, Beyotime Institute of Biotechnology, Haimen, China) was added to each well and incubated at 37°C for 4 h. Then, the optical density of each well was recorded using a microplate reader (Bio-Tek, Winooski, VT, USA) at 450 nm.

Apoptotic cells were identified by performing the morphological staining of nuclei. A549 cells were fixed in 4% paraformaldehyde, stained with DAPI for 10 min and photographed using a fluorescence microscope (Leica, Weztlar, Germany). Apoptotic cells were identified based on their typical morphological appearance, including chromatin condensation and nuclear fragmentation.

Cells were washed with PBS three times, fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% Triton X-100 in PBS for 20 min and then blocked in PBS with 1% bovine serum albumin (BSA) for 30 min. The cells were incubated with the appropriate primary antibodies and then stained with Alexa Fluor 633-labeled goat anti-rabbit IgG and Alexa Fluor 488-labeled goat anti-mouse IgG. Cell nuclei were stained with 5 μg/ml DAPI (Sigma-Aldrich). Following staining, cover slips were analyzed using a Leica SP8 confocal microscope.

Cells were lysed, and total RNA was extracted using TRIzol reagent. cDNA was made from total RNA using oligo (dT) primers with a RevertAid First Strand cDNA Synthesis Kit (K1621, Fermentas, Waltham, MA, USA) according to the manufacturer's instructions. qPCR was performed using the TB Green and StepOnePlus PCR system (Applied Biosystems). The following conditions were used for amplification of fragments: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and 60°C for 30 s. The analysis of the Ct values of cells was normalised to GAPDH for each sample. The normalised values (ΔCt) of all samples were compared with the control in each group (ΔΔCt). The results are expressed as 2^−ΔΔCt, and the resulting data derive from at least three independent experiments. The gene-specific primer sequences used are listed in Table S1.

Cells were lysed on ice for 30 min in lysis buffer containing 1% NP40, 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM EDTA and protease inhibitor cocktail. After centrifugation, supernatants were incubated with anti-Flag sepharose affinity gel (A2220, Sigma-Aldrich) or with Protein A/G PLUS-Agarose beads (sc-2003, Santa Cruz Biotechnology) at 4°C overnight. After five washes in wash buffer [1% NP40, 300 mM NaCl, 10 mM Tris-HCl (pH 8.0), 10% glycerol and 1 mM EDTA], the immunoprecipitants were analyzed by immunoblotting. For western blot analysis, equal amounts of cell lysates and immunoprecipitants were resolved on a 10-12% SDS-PAGE and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA/TBS plus 0.1% Tween 20 (TBST) at room temperature for 2 h, and incubated with primary antibodies at 4°C overnight. The membranes were then incubated with a secondary antibody for 1 h at room temperature followed by three 10-min washes in TBST. The bands were visualized by enhanced chemiluminescence (32106, Thermo Fisher Scientific).

The intensity of the western blot results was analyzed by densitometry using ImageJ Launcher software (National Institutes of Health) for quantification. All experiments were repeated for at least three independent batches, and each batch was performed for at least three biological replicates. The averages and standard errors of all the data shown were calculated from three biological replicates of one batch experiment. Data are expressed as mean±s.d. Statistical analyses were performed using Prism 5 software (GraphPad Prism, Version X; La Jolla, CA, USA). Statistics were calculated using Student's t-test. P<0.05 was considered statistically significant.

The peer review history is available online at https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.259018.