Fig 2: Targeting circ_0000654 affects tumor growth in vivo. (a) Growth curves of the xenograft tumors generated by subcutaneous implantation of 2 × 106 sh-circ_0000654- or sh-NC-transduced ECA-109 cells (n = 6 each group).Representative images and mean weight (b), qRT-PCR analysis of circ_0000654 and miR-375 (c), immunohistochemistry of E2F3, BCL-2 and c-Myc staining (d) of the xenograft tumors formed by subcutaneous implantation of 2 × 106 sh-circ_0000654- or sh-NC-transduced ECA-109 cells on day 28 after cell injection (n = 6 each group). ***p < 0.001

Fig 3: TMEM119 silencing induced SGC-7901 cell apoptosis. After transfection of SGC-7901 cells with TMEM119-siRNA (siTMEM119), cell apoptosis was measured by (A and B) flow cytometry assay and the expression of Bcl-2, Bax as well as caspase-3 was measured by (C and D) western blot analysis. **P<0.01, compared to SGC-7901. TMEM119, transmembrane protein 119.

Fig 4: BDA-366 does neither directly activate Bax nor indirectly via Bcl-2.a Representative plot of the liposome permeabilisation assay with BDA-366 and different Bcl-2-family members. Liposomes (0.04 mg/mL) encapsulating ANTS and DPX were incubated with BDA-366 (0.1–10 µM), Bim (10 nM) and Bax (100 nM) in the presence or absence of Bcl-xL (40 nM). Liposomes incubated with Bax (100 nM) and Bim (10 nM) were used as positive control, while DMSO (1%) served as the vehicle control. Fluorescent intensity of ANTS (excitation = 355 nm and emission = 520 nm) was measured during 120 min, whereas Bax (100 nM) was added at t0. b Results obtained from endpoint measurements are presented as mean ± SD of five independent experiments. Significance, compared to control condition with only Bax, was determined using a one-way Anova with post hoc Dunn’s multiple comparisons test. c Analysis of permeabilisation of liposomes incubated with 10 nM Bim and 100 nM Bax and increasing concentrations of Bcl-2 (N = 3). d Representative plot of the liposome permeabilisation assay with BDA-366 and Bcl-2. Liposomes encapsulating ANTS and DPX and were incubated with 1 µM BDA-366; 100 nM of Bcl-2 and Bax (100 nM). Fluorescent intensity of ANTS (excitation = 355 nm and emission = 520 nm) was measured during 120 min. Liposomes incubated with Bax (100 nM) and Bim (10 nM) were used as positive control. e Similar experiment as in Panel A using varying concentration of BDA-366 or ABT-199 (ranging from 0 to 10 µM), Bcl-2?TMD (100 nM), Bax (100 nM), and Bim (20 nM). Data are presented as averages ± SD of three independent experiments at 10 min intervals for 180 min. f Results obtained from endpoint measurements are presented as mean ± SD of three independent experiments. Significance, compared to control condition with only Bax, was determined using a one-way Anova with post hoc Dunnett’s multiple comparisons test. g Thermal denaturation curves of Bcl-2 (15–85 °C) were obtained by monitoring ellipticity at 222 nm, by far-UV CD, while heating the protein samples [15 µM; 250 µL; 5 mM MOPS, pH: 7.5; 5 mM NaCl; 0.5% DMSO; BDA-366 (50 µM) or ABT-737 (5 µM) as indicated] at 1 °C/min. A representative experiment is shown. h Quantitative analysis of Tmapp of purified Bcl-2, in the presence or not of drugs (as indicated), obtained from experiments similar to those presented in (g). Data presented here are averages ± SD of independent experiments (N > 3). Statistical significance was determined with a one-way ANOVA with a Tukey’s multiple comparisons test comparing apoprotein with BDA-366 or ABT-737.

Fig 5: miR-210 mitigates the hypoxia-driven decrease in MCL1 levels in the mitochondria through the inhibition of GSK3ß kinase activity. (A–F) Quantitative ELISA immunoassays determining the abundance of the BCL2 family of antiapoptotic proteins, BCL-XL (A,B), BCL2 (C,D), and MCL1 (E,F), in the mitochondrial fractions (A,C,E) and the cytosolic fractions (B,D,F). Data are expressed as experimental blank-corrected absorbances (O.D) measured at ?450 (450 nm) normalized to fold-change values. Quantitative ELISA immunoassays determining the abundance of BCL-XL, BCL2, and MCL1 in the whole-cell fractions are reported in Supplementary Figure S5A–C. miR-210 expression levels in the respective cell lysates were determined by the miR-210 hybridization immunoassay (as described in Section 4.3) and are reported in Supplementary Figure S1A. The validation of the ectopic expression of the HA-tagged ca-GSK3ß-S9A mutant in the pertinent experimental groups was performed by ELISA immunoassay and is reported in Supplementary Figure S1C. GSK3ß kinase activity (as described in Section 4.4) was measured in all experimental groups to corroborate and validate the translative effects of the ectopic expression of the ca-GSK3ß-S9A mutant (reported in Supplementary Figure S1E). All data are expressed as mean ± S.D fold-change values from three technical replicates for each of the four biological replicates belonging to each experimental group (n = 4). * p = 0.05; ** p = 0.01; *** p = 0.001; **** p = 0.0001; ns: not significant (p > 0.05). OE: miR-210 overexpression; O.D: optical density; S.D: standard deviation.