Fig 2: BMPR2 regulation of mitochondrial Ca2+transport through the MCU effects mitochondrial bioenergetics and cell survival. A Western blot analysis of Jurkat and MDA-MB-231 WT and MCU KO cells. B Flow cytometry analysis of the fluorescence of Rhod-2AM of Jurkat WT and MCU KO cells treated with JL189 1.25 µM for 16 h. The graph represents the mean fluorescence of 4 independent biological replicates presented as percent change from its vehicle DMSO control. C Flow cytometry analysis of the fluorescence of Rhod-2AM of MDA-MB-231 WT and MCU KO cells treated with JL189 2.5 µM for 5 h. The graph represents the mean fluorescence of 2 independent biological replicates presented as percent change from its vehicle DMSO control. D-E Cell counts of (D) Jurkat and (E) MDA-231 WT and MCU KO cells treated with JL189 2.5 µM for 24 and/or 48 h. The graphs represent the mean percent number of dead cells of at least 4 independent biological replicates. F Flow cytometry analysis of the fluorescence of MitoTracker Green of MDA-MB-231 WT and MCU KO cells treated with JL189 2.5 µM for 16 h. The graph shows with mean fluorescence of 5 independent biological replicates presented as percent change from its vehicle DMSO control. G, I Western blot analysis of THP1 and H1299 transfected with TAB1 cDNA for 24 h done in duplicate. Graphs represent normalized TFAM1 and cytochrome b expression as the mean percent change from control of (G) 3 and (I) 2 independent biological replicates. H, J Western blot of THP1 and H1299 cells treated with 5Z-7-OXO for 2 h. Graphs represent normalized TFAM1 and cytochrome b expression as the mean percent change from control. H 3 independent biological replicates for TFAM1 and cytochrome b. J 3 independent biological replicates for TFAM1 and 2 independent biological replicates for cytochrome b. K Western blot of H1299 cells transfected with control or MCU siRNA for 48 h then treated with 5Z-7-OXO for 2 h. Graph represents the mean normalized TFAM1 expression of 3 independent biological replicates. L Western blot of H1299 tumor xenografts treated with vehicle or JL189 for 3 weeks. Graphs represent normalized values of 6 independent biological replicates. M Western blot analysis of immunoprecipitated (IP) TAB1-Flag or whole cell lysates of Calu-1 cells transfected with insertless cDNA or TAB1-Flag plasmids. Upper immunoblots show short film exposure and lower blot longer film exposure. N-O CellTiter-Glow assay was used to measure ATP levels. N Cells were treated with DMSO or JL189 2.5 µM for 4 h, or 1.5 µM oligomycin A for 50 min. O Cells were treated with DMSO or JL189 2.5 µM for 4 h. Graphs represent 3 independent biological replicates presented as the percent change from control. Graphs show standard errors and p values represent the paired Student’s t test, assuming unequal variances

Fig 3: BMPR2i increases TCA cycle intermediates, mitochondrial respiration, and mitochondrial ATP production in NSCLC cell lines. A Western blot of A549 WT and A549 BMPR2 KO cells. B Metabolomic studies of A549 WT and A549 BMPR2 KO cells. C Mitochondrial respiration was determined using the Seahorse Analyzer of A549 WT and A549 BMPR2 KO cells. D Metabolomics studies of A549 and H1299 cells treated with JL189 2.5 µM for 24 h. E-F Mitochondrial respiration was determined using the Seahorse Analyzer of A549 and H1299 cells treated with JL189 2.5 µM for 24 h. G-I CellTiter-Glow assay was used to measure ATP levels. G Cells treated with DMSO or oligomycin A 1.5 µM for 30 min followed by DMSO or JL189 2.5 µM for 2 h. H Cells treated with DMSO or 10 µM oligomycin A for 50 min followed by DMSO or JL189 2.5 µM for 2 h. I Cells were treated with DMSO or JL189 1.5 µM for 4 h. Graphs represent the mean of 3 independent biological replicates reported as the standard error. p values represent the paired Student’s t test, assuming unequal variances

Fig 4: BMPR2 regulates TAK1-d splice variant to mediate mitochondrial Ca2+ transport. A Western blot analysis of NSCLC and leukemia cell lines. B Western blot analysis of cells transfected with TAB1 cDNA or control vector after 24 h. C-D Western blot analysis of cells treated with JL189 for (C) 2 h or for (D) 4 h. E Western blot analysis of Calu-1 cells transfected with BMPR2 and control siRNA for 48 h. Graphs depict changes in the mean phosphorylation of TAK1 Thr184/Thr187 and TAK1 Ser412 expression of 3 independent biological replicates. F Western blot analysis of A549 WT and BMPR2 KO cells. G-H Western blot analysis of cells treated with BMPR1 receptor inhibitors. THP1 cells were treated for 4 h. Graphs represent normalized pTAK1 expression of (G) 3 independent biological replicates and (H) 4 independent biological replicates. I Western blot analysis demonstrating TAK1 splice variants using an antibody recognizing the N-terminal end of TAK1. The top is the immunoblot with a short film exposure and bottom immunoblot is with longer film exposure. J Western blot of THP1 cells treated with JL189 for 6 h. Graph represents normalized values of 2 independent biological replicates. K Western blot of cells transfected with TAB1 or control plasmid vectors for 24 h. Graph represents normalized values of 2 independent biological replicates. L-N Flow cytometry analysis of the fluorescence of Rhod-2AM of cells treated with TAK1 inhibitor 5Z-7-OXO 2 µM or 4 µM for 2 h. Graphs represent mean fluorescence of 3 independent biological replicates. O Western blot analysis of cells transfected with control or TAB1 siRNA for 48 h. Graph represents the mean percent change compared to control in normalized pTAK1 expression of 3 independent biological replicates. P-Q Flow cytometry analysis of the fluorescence of Rhod-2AM of cells transfected with TAB1 siRNA or (M) TAB1 cDNA. The graphs represent the mean Rhod-2AM fluorescence intensity of 3 (TAB1 siRNA) and 4 (TAB1 cDNA) independent biological replicates. Graphs show standard errors and p values represent the paired Student’s t test, assuming unequal variances