Fig 2: Diva silencing increases the proportion of apoptotic cells. a Flow cytometry performed on live PC12 cells within 1 hr of Annexin V staining. PC12 cells were stained 48 hrs after transfection with negative control and Diva siRNA. b Flow cytometry performed on live PC12 cells within 1 hr of PI staining. PC12 cells were stained 48 hrs after transfection with negative control and Diva siRNA. c Flow cytometry result of Annexin V stained negative control siRNA and Diva siRNA transfected BMSCs. d Flow cytometry result of PI stained negative control siRNA and Diva siRNA transfected BMSCs. Annexin V and PI staining were performed on live BMSCs 48 hrs after transfection. e–f Graphs showing the relative increase in Diva siRNA transfected PC12 cells that had taken up the Annexin V and PI stains respectively. g–h Graphs showing the relative increase in Diva siRNA transfected BMSCs that had taken up the Annexin V and PI stains respectively. Results were statistically significant with p-values <0.01.

Fig 3: Loss of Diva decreases cell number and viability. a PC12 was transfected with both negative control siRNA and Diva siRNA to assess PC12 for changes in cell attachment to plate surface following Diva silencing. The top panel shows cells 48 hrs after transfection while the bottom panel shows cells 72 hrs after transfection. b Cell attachment images of BMSCs 48 hrs and 72 hrs after silencing. Bar = 100 µm. c MTS assay was used to monitor the changes in PC12 cell number following transfection with negative control siRNA and Diva siRNA. Graph showing the percentage of viable PC12 cells in relation to the negative control after Diva silencing for 48 hrs and 72 hrs. Neg and Diva represent negative control siRNA and Diva siRNA2 respectively. d Graph showing the percentage of viable BMSCs in relation to the negative control after Diva silencing for 48 hrs and 72 hrs. Results were statistically significant with p-values <0.01.

Fig 4: Bcl2l10-knockdown significantly decreases the protein expression levels of SDHD and IDH1. (A) SKOV3 and A2780 cells were transfected with siNC or siBcl2l10 and harvested 48 h after transfection. The protein expression levels of BCL2L10, SDHD, IDH1 and FH were measured by western blot analysis using β-actin as an internal control gene. (B) Protein signals of BCL2L10, SDHD, IDH1 and FH were measured and normalized to the corresponding β-actin signals. The relative expression levels of proteins were calculated by comparing all the normalized signals to that of the negative control group. All the experiments were repeated at least three times, and the data are expressed as the mean ± SEM. *P<0.05 and **P<0.01 vs. siNC. si, small interfering RNA; NC, negative control; Bcl2l10, Bcl-2-like-10; SDHD, succinate dehydrogenase complex subunit D; IDH1, isocitrate dehydrogenase 1; FH, fumarate hydratase.

Fig 5: IRBIT and Bcl2l10 cooperate to regulate IP3R activity.(A) Representative Ca2+ response curve of Fura-2-loaded IRBIT KO MEF cells stimulated with 1 µM ATP at the indicated times. Cells were transfected with empty vector or with a plasmid expressing either FLAG-Bcl2l10 or FLAG-IRBIT alone, or FLAG-Bcl2l10 and FLAG-IRBIT together. Basal Fura-2 F340 nm/F380 nm = 0.61 ± 0.01, 0.59 ± 0.01, 0.59 ± 0.01 and 0.58 ± 0.01 for empty vector, Bcl2l10, IRBIT and Bcl2l10+IRBIT, respectively. (B) Bar graph showing the mean amplitude (±SEM) of the ATP-induced Ca2+ peak (n: number of cells analyzed from five independent experiments). (C) Western blot of GST-pulldown performed with GST or GST-IP3BD on lysates of HeLa cells expressing HA-IRBIT alone or in combination with FLAG-Bcl2l10. Quantification was performed from three independent experiments. (D) Western blot of GST-pulldown performed with GST-IP3BD on recombinant Bcl2l10 alone or in combination with recombinant IRBIT produced in Sf9 cells. Quantification was performed from three independent experiments.(E) Western blot of GST-pulldown performed with GST-IP3BD on lysates of HeLa cells expressing HA-IRBIT alone or in combination with FLAG-Bcl2l10 in the presence of 0, 1 or 10 µM IP3. Quantification was performed from three independent experiments. *p<0.05, **p<0.01, ***p<0.001.DOI: http://dx.doi.org/10.7554/eLife.19896.003