Fig 1: Identification of candidate targets of CLPTM1L by iTRAQ-based analysis. a Expression levels of CLPTM1L in lung adenocarcinoma compared with normal lung tissues obtained from public databases (Selamat dataset). Student’s t test; ** P < 0.01. b Proteins upregulated or downregulated by CLPTM1L in A549 cells exposed to IR were identified by iTRAQ-based analysis based on the P-value (P < 0.05). c–d Distribution of mass or isoelectric point of the 1237 proteins identified. e–f Analysis of parallelism between CLPTM1L overexpressing groups or control groups from iTRAQ-based analysis. g Volcano plot showing 233 CLPTM1L-modulated genes among the 1237 genes identified based on fold changes in expression [log10(FC) < - 0.14 or log10(FC) > 0.14]
Fig 2: CLPTM1L induces radioresistance of NSCLC cells by coactivating ERß. a Effect of CLPTM1L and ERß on A549 and H460 cell proliferation after exposure to 0 or 4 Gy of IR as determined by the EdU assay. b–c MTT assays of the effect of ERß/pcDNA-CLPTM1L-m (CLPTM1L containing a mutated LXXLL motif) on CLPTM1L-modulated radiosensitivity in A549 and H460 cells were performed after 3 days of IR. d–e A549/H460 cells pretreated with CLPTM1L siRNA/pcDNA-CLPTM1L and pcDNA-ERß/ERß siRNA/pcDNA-CLPTM1L-m were exposed to IR (0, 2, 4, and 8 Gy) and analyzed by clonogenic cell survival assay after 12 days of IR. The data presented are from three independent experiments; Student’s t test; * P < 0.05; ** P < 0.01; NS, not significant
Fig 3: Silencing of CLPTM1L sensitizes xenograft NSCLC tumors to IR in an animal model. a Mice (n = 40) bearing NSCLC xenograft tumors developed from H460 or A549 cells transfected with control or CLPTM1L shRNA were treated with or without local IR, as illustrated in the diagram. b Growth of tumors generated by transplantation of NSCLC cells. c Images of dissected tumors from nude mice (n = 5/group). d Weight of resected xenograft tumors in nude mice. e Representative images (400× magnification) of H460 and A549 xenografts stained for CLPTM1L and Ki-67. f Real-time PCR analysis of the mRNA levels of CDC25A, c-Jun, and BCL2 in H460 and A549 xenografts. Statistically significant differences are indicated. NS, not significant. Student’s t test; * P < 0.05; ** P < 0.01. g The model shows that IR can increase the expression of CLPTM1L, and CLPTM1L upregulates the expression of ERß-induced genes by coactivating ERß via the LXXLL motif, leading to the radioresistance of NSCLC cells
Fig 4: CLPTM1L activates the promoter of ERß-induced genes by stimulating ERß in NSCLC cells. a–i Cells were transfected with relative plasmids and siRNAs, exposed to 4 Gy IR, followed by luciferase reporter gene assays. a ERE-LUC activity was examined using luciferase reporter gene assays in A549 and H460 cells transfected with pcDNA-CLPTM1L plasmids (E2, also called 17ß-estradiol, was used as the positive control). b ERE-LUC activity was examined using luciferase reporter gene assays in A549 cells transfected with pcDNA-CLPTM1L plasmids or CLPTM1L siRNA. c ERE-LUC activity was determined using luciferase reporter gene assays in H460 cells treated with various doses of pcDNA-CLPTM1L plasmids. d The mutation of the ERE is shown in the ERE-LUC construct. e Luciferase reporter gene assays in A549 and H460 cells transfected with ERE-LUC constructs with wild-type or mutant ERß-binding sites (E2 was used as the positive control). f ERE-LUC activity was detected by luciferase reporter gene assays in A549 and H460 cells transfected with pcDNA-CLPTM1L and ERß siRNAs. g The effect of CLPTM1L on ERE-LUC activity was tested by luciferase reporter gene assays in ER-negative cells (MDA-MB-231) transfected with or without pcDNA-ERß/pcDNA-ERa. NS, not significant. h ERE-LUC activity was detected by luciferase reporter gene assays in A549 and H460 cells transfected with pcDNA-ERß and CLPTM1L siRNAs. i ERE-LUC activity was examined by luciferase reporter gene assays in A549 and H460 cells transfected with pcDNA-CLPTM1L and/or pcDNA-ERß plasmids. All experiments were repeated at least three times. Student’s t test; * P < 0.05; ** P < 0.01; *** P < 0.001
Fig 5: CLPTM1L upregulates the levels of CDC25A, c-Jun, and BCL2 through ERß in NSCLC cells. a Real-time PCR analysis of the mRNA levels of CLPTM1L, CDC25A, c-Jun, and BCL2 in A549 cells exposed to different doses of ?-ray IR. b Western blot analysis of the protein levels of these genes in A549 cells. c Western blot analysis of the protein levels of these genes in H460 cells exposed to 4 Gy IR. d–e Real-time PCR and western blot analysis of the mRNA and protein levels of CLPTM1L, ERß, CDC25A, c-Jun, and BCL2 in A549 cells treated with IR and siRNAs. f–g Real-time PCR and western blot analysis of the expression of these genes in H460 cells treated with the indicated plasmids/siRNAs and 4 Gy IR. All experiments were repeated at least three times. Statistically significant differences are indicated. Student’s t test; * P < 0.05; ** P < 0.01
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