Fig 2: Effect of CIAPIN1 upregulation on NSCLC A549 cells' metastatic potential was measured. (a) Representative images of cells' migration and invasion were taken. (b) The relative cells' migration and invasion after CIAPIN1 upregulation were measured. (c, d) The mRNA expression and the protein levels of MMP2, MMP9, and MMP14 were detected by real-time quantitative PCR and western blotting. (e, f) The mRNA expression and the protein levels of snail, slug, twist, zeb1, and E-cadherin were detected by real-time quantitative PCR and western blotting. (g) Expressions of cell cycle- and apoptosis-associated proteins were detected by western blotting. The expressions of cyclin D1, cyclin D2, cyclin D3, cyclin E, cdk2, and cdk4 were detected in A549 cells with CIAPIN1 overexpression. The expressions of Bax, Bid, Bim, Bcl-2, and Mcl-1 were detected in A549 cells with CIAPIN1 overexpression. GAPDH was used as an internal control. Each value indicated the mean ± SD of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, compared with control.

Fig 3: IS21 reduces the viability of solid tumor cell lines and alters the interactions between Bcl-2 and Bcl-xL with their targets.Analysis of cell viability of A melanoma and B ovarian cancer cell lines treated with increasing concentrations of IS21 for respectively 72 h and 5 days. Results are reported as “viability of treated cells/viability of control cells (Ctrl)” × 100, and as mean ± SD of three independent experiments. C Western blot analysis of BAX and BAK protein levels in A375 melanoma cell lines control (si-K), silenced for BAX (si-BAX), BAK (si-BAK), or both proteins (si-BAX + BAK). The same filter was used first for BAK and then for BAX. Analysis of D cell viability and E relative IC50 values of A375 melanoma cells silenced as reported in C and treated as reported in A. p-value was calculated between control and cells silenced for BAX, BAK or both, *p ≤ 0.05. F–H Western blot analysis of Beclin-1, anti-apoptotic (Bcl-2, Bcl-xL, Mcl-1), and pro-apoptotic (BAX, BAK, BIM, NOXA) proteins after immunoprecipitation of A375 melanoma cells with F Bcl-2, G Bcl-xL, or H Mcl-1 antibodies after treatment with 20 μM IS21 for 48 h. Control cells (Ctrl). C, F–H Reported western blot images are representative of two independent experiments with similar results. β-actin, α-tubulin, and Hsp72/73 are shown as loading and transferring control, molecular weights are expressed in kilodalton (kDa).

Fig 4: Effects of RT on the expression of the apoptotic-related proteins: Mcl-1, Bcl-2, and Bax in the NSCLC cell line (H460) and patient-derived primary cancer cell lines (ELC12, ELC16, ELC17, and ELC20). (a) H460, ELC12, and ELC16 cell lines were seeded and treated with 0–1 μM of RT for 24 h. Then, immunofluorescence analysis was performed using an antibody against Mcl-1. The Alexa Fluor 488 conjugated secondary antibody and Hoechst 33342 were added and the images were visualized under a fluorescence microscope. (b) ELC12, ELC16, ELC17, and ELC20 cell lines were seeded and treated with 0–1 μM of RT for 24 h. Western blot analysis was performed to detect the Mcl-1, Bcl-2, and Bax protein levels. The blots were reprobed with β-actin to confirm an equal loading of each of the protein samples and densitometry was used to calculate the relative protein levels. Densitometric values of protein expression levels were presented as the fold changes relative to β-actin. (c) H460 cells were seeded and treated with 0–1 μM of RT, TM-(–)-45, TM-(–)-18, TM-(–)-4a, TM-(–)-52, and TM-(–)-55 for 24 h. After that, Western blot analysis was performed to detect the Mcl-1, Bcl-2, and Bax protein levels. Data represent the mean ± SEM (n = 3) (* 0.01 ≤ p < 0.05, ** p < 0.01, compared with the untreated control).

Fig 5: CD157 promotes survival of leukemic blasts through Mcl-1 upregulation. (A) CD157-high (red line) or CD157-low (blue line) U937 cells were treated for 24 h with increasing concentrations of S63845, then cell viability was determined by Presto Blue assays. Data represents the percentage of live cells compared to control cells treated with vehicle and are the mean ± SEM of three experiments performed in six replicates. (B) Western blot analysis of PARP-1, Mcl-1 and cleaved Caspase-3 in CD157-high or CD157-low U937 cells treated with S63845 for 24, 48 or 72 h. β-Tubulin was used as loading control. Uncropped blot images are provided in Supplementary Fig. S11. (C) CD157-high or CD157-low U937 cells were treated for up to 72 h with S63845. Cell apoptosis was measured by flow cytometry analysis after AnnexinV/PI staining. Results are the mean ± SEM of three experiments. *p < 0.05, two-way ANOVA with Sidak’s multiple comparison test. (D) U937/CD157-high or U937/CD157-low cells were treated with S63845 for 24 h, then lysed and subjected to immunoprecipitation with anti-Mcl-1 or anti-Bim antibody, respectively. The interactions with the indicated proteins were analyzed by immunoblotting. β-Tubulin was used as loading control. One representative Western blot is shown (n = 3). The whole cell lysates (WL) show the protein expression levels. Uncropped blot images are provided in Supplementary Fig. S11.