Fig 2: Effects of ONC201 on mitochondrial functions in NB cells: (A) The relative protein levels of ClpP, ClpX, SDHB, and NDUFS1 were determined by Western blot and quantified by Image Pro-plus analysis software. (B,C) OCR in ONC201-treated cells were detected by the Seahorse XF24 analyzer in the absence or presence of oligomycin (1 µM), FCCP (0.5 µM), and rotenone (1 µM) by counting 1 × 105 cells. (D,E) Profiling of ECAR in control and ONC201-treated cells was measured by the Seahorse XF24 analyzer. (F) Cells were labeled with TMRM (100 nM) to examine mitochondrial membrane potential and analyzed by flow cytometry. (G) Total cellular ATP levels were measured using the luciferase-based luminescence assay kit. All data were obtained from three independent experiments, and are expressed as mean ± SD. * p < 0.05.

Fig 3: Knockdown of APOBEC3A (A3A) reduces C>U RNA editing in M1 macrophages(a) A3A and APOBEC3G (A3G) gene expression in M1 macrophages that were transfected with a nonspecific (Ctrl.) or either one (1, 2) or equimolar mix (1+2) of two A3A-specific siRNAs at 100 nM concentration. Gene expression measurements are normalized to that for ACTB. (b) Immunoblot for A3A protein (23 kDa) of whole-cell lysates (10 µg protein) of two of each set of three replicate transfectants. Nonspecific signals are indicated by an asterisk (*). The signal for calnexin, a house-keeping protein, indicates total protein. (c) SDHB c.136C>U RNA-editing levels in the siRNA transfectants, which are determined by RT–qPCR. (d) Sanger sequence chromatogram traces of amplified cDNA fragments, indicating reduced site-specific RNA editing for five other genes in A3A-specific siRNA 1 compared with Ctrl. transfectants. Mean and range (n=3) are shown for a and c.

Fig 4: APOBEC3A induces C>U RNA editing in 293T transfectants.(a) Immunoblots showing APOBEC3A (A3A), APOBEC3G (A3G) and CDA proteins in whole-cell lysates (20 µg protein) of 293T cells transiently transfected with an empty vector (Ctrl., control) or DNA constructs for expression of A3A, A3G or CDA proteins. (b) SDHB c.136C>U RNA editing in the 293T transfectants, which were optionally treated with hypoxia and/or 600 U ml-1 type I IFN (IFN1). Mean and range for n=3 are shown. (c) Estimation of site-specific C>U RNA editing by Sanger sequencing of RT–PCR products for 30 genes in the transfectants (n=1). The detection limit for editing (5% level) is indicated. Samples without detectable editing were assigned a value of 3.8%. Chromatograms for 19 genes are shown in Supplementary Fig. 5. Chromatograms of good quality could not be obtained for C1QA and TMEM179B for the A3G and CDA transfectants, and for the GPR160 site for the normoxic A3A transfectant. (d) Chromatograms of genomic DNA (gDNA) and cDNA PCR products of normoxic A3A transfectants, indicating C>U RNA editing without C>T genomic change at positions marked with * for ASCC2, SDHB and TMEM109. Immunoblots showing ASCC2, SDHB and TMEM109 proteins in whole-cell lysates (20 µg protein) of control or A3A transfectants on the right indicate reduced protein expression in association with A3A-induced stop codons in RNA. Only a single band of signal, which corresponded to a protein of full length, was seen in all three immunoblots.

Fig 5: SQR inhibition via Qp site SDHC-mutant introduction sensitizes MM to venetoclax.a SQR, b SDH and c CS activities were determined in KMS11 and L363, SDHCKO cells expressing SDHC-WT or SDHC-R72C mutant constructs. n = 4 independent experiments except L363 SQR and SDH activity (n = 3). Data are presented as mean values ± SEM. Adjusted p values are calculated using a two-way ANOVA with post-hoc Sidak’s multiple comparisons test. d SDHC-WT and SDHC-R72C mutant-expressing cells ± 0.5 µM venetoclax (24 h) were evaluated for viability by AnnexinV/DAPI flow cytometric staining, demonstrating increased sensitivity of SDHC-R72C cells to venetoclax. n = 4 independent experiments. Adjusted p values are calculated using a two-way ANOVA with post-hoc Tukey’s multiple comparisons test. e Spare respiratory capacity determined in the indicated SDHC-WT and SDHC-R72C-mutant-expressing cells demonstrated a net reduction in SRC upon introduction of the mutant. n = 6 replicate Seahorse wells except KMS11 SDHC-R72C (n = 4). Data are presented as mean values ± SEM. Adjusted p values are calculated using a two-way ANOVA with post-hoc Sidak’s multiple comparisons test. **** denotes p value < 0.0001. Source data are provided as a source data file.