Fig 2: Increased mitochondrial fission affects apoptosis of HCC cells through coordinately regulating the NFKB and TP53 pathways. (A) Western blot analyses for protein levels of DNM1L, apoptosis-related molecules BCL2, BCL2L1, BID, TP53 and RELA in HCC cells with treatment as indicated. LMNB1 (a nuclear envelope marker) and ACTB were used as loading controls in the nuclear and cytoplasmic fractions, respectively. (B) Western blot analyses for protein levels of DNM1L and apoptosis-related molecules in whole-cells or RELA in cytoplasm and nucleus of SMMC7721 cells transiently transfected with the DNM1L expression vector and then followed by treatment with the NFKB inhibitor Bay11-7082 (12.5 µM) for 12 h. (C) Cell apoptosis was respectively evaluated in SMMC7721 cells with treatment as indicated. (D) Western blot analyses for protein levels of DNM1L, TP53 and apoptosis-related molecules in SMMC7721 cells transiently tansfected with DNM1L and/or TP53 expression vectors as indicated. (E) Cell apoptosis was evaluated in SMMC7721 cells tansfected with different expression vectors as indicated. (F) Western blot analyses for protein levels of cleaved CASP9 and cleaved CASP3 in HCC cells treated with obatoclax (5 µM) for 12 h before cell harvest. (G) The correlation was evaluated between the protein expression levels of DNM1L and TP53 or nuclear RELA in 128 HCC tissues based on IHC staining.

Fig 3: VHL-based PROTAC-BCL-XL DT2216 is also active in killing BCL-XL-dependent GC cells. (A) Immunoblotting analysis of E3 ligase MDM2 and VHL expression in both GC and blood cancer cell lines. (B) Minimal expression of VHL and MDM2 in human platelets. (C) Sensitivity of human GC cell lines to the VHL-based PROTAC-BCL-XL DT2216. The viability of 23 132/87, SNU216 and NCI-N87 to DT2216 after treatment with 0–10 µM of DT2216 for 24–96 h was determined using the CellTiter-Glo assay. (D) BAX/BAK-dependent killing of GC cells by DT2216. The viability of WT 23132/87 cells or the BAX-/-BAK-/- subclones after treatment with 0–10 µM of DT2216 was determined at 72 h using the CellTiter-Glo assay. (E) Protein levels of BCL-XL, MCL1 and BCL2 upon DT2216 treatment. BAX-/-BAK-/- SNU216 cells were treated with 0–10 µM of DT2216 for 96 h and the protein levels of BCL-XL, BCL2 and MCL1 were determined. (F) DT2216 induces degradation of BCL-XL in a time-dependent manner. (G) Proteasome inhibition blocks the BCL-XL degradation by DT2216. Immunoblotting analyses of BCL-XL protein levels in BAX-/-BAK-/- SNU216 cells after they were either left untreated or pretreated with the proteasome inhibitor MG132 (5 µM) for 1 h, and then treated with or without DT2216 for 24 h. The immunoblots in (A,B,E–G) are representatives of =2 independent experiments; data in (C) and (D) represent the means ± SD of =3 independent experiments.

Fig 4: A subset of GC cell lines are susceptible to selective BCL-XL inhibitors. (A) Sensitivity of human GC cell lines to the small molecular inhibitors of BCL-XL. The sensitivity of the 7 human GC lines to A1155463, A1331852 and ABT-263 was determined using the CellTiter-Glo assay after culturing in 0–10 µM of the indicated compounds for 24 h. A discrete heat map representation of the mean ± SD of IC50s from 3 independent experiments is shown in this panel. Red represents potent killing (IC50 < 0.1 µM), whereas blue indicates resistance (IC50 > 10 µM). (B) BAX/BAK-dependent killing of GC cells by these selective BCL-XL inhibitors. Top panel: loss of BAX and BAK expression in the engineered knock-out clones was confirmed by Sanger DNA sequencing and western blotting. Bottom panel: the viability of WT 23132/87 cells or the BAX-/-BAK-/- subclones after treatment with 0–10 µM of the indicated compounds was determined using the CellTiter-Glo assay. (C) Genetic deleting BCL-XL rapidly induces cell death in 23 132/87 cells. The viability of 23 132/87 cells 0–120 h after addition of doxocycline (DOX) to induce expression of guide RNAs (sgRNAs) that target BCL-XL was determined with the CellTiter-Glo assay. Two sgRNAs targeting BCL-XL were tested. (D) ABT-263 is less effective at releasing BIM from BCL-XL than A1331852. Equivalent lysates prepared from 23 132/87 cells treated with the indicated concentrations of ABT-263 or A1331852 in the presence of 20 µM QVD were immunoprecipitated with the BCL-XL or BCL2 antibody. The amount of BIM associated with BCL-XL upon indicated treatments was determined. Total amount of BIM and ß-actin in the whole cell lysates served as the loading controls. Data in (A–C) represent the means ± SD of =3 independent experiments; data shown in (D) are from a representative of two experiments.

Fig 5: Biomarkers predicting the dependence of GC cells on BCL-XL. (A) BCL2L1 CNVs, BCL-XL mRNA and BCL-XL protein levels in the GC cell lines used in this study. Top: error bars indicate the Poisson 95% confidence intervals for each determination. The dashed line indicates the ddPCR threshold cut-off of 3.0 copies for calling a sample BCL2L1-amplified. Middle: relative BCL-XL mRNA expression normalized to GADPH. Bottom: BCL-XL protein expression was quantified by western blotting and densitometric analysis using ImageJ. Relative BCL-XL protein levels normalized to SNU216 were used for subsequent correlation analysis. (B) Simple linear regression of BCL2L1 CNVs, BCL-XL mRNA or BCL-XL protein levels. (C) Simple linear regression between IC50s to A1331852 and BCL2L1 CNV, BCL-XL mRNA or BCL-XL protein levels. (D) Protein levels of other BCL2 family members in the GC cell lines used in this study. (E) Simple linear regression between IC50s to A1331852 and BCL2, MCL1, BIM or NOXA protein levels.