Fig 2: 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 3: BMPR2i in NSLC lung cancer cells increases mtCa2+levels. A-E Flow cytometry analysis of the fluorescence of Rhod-2AM-DAPI (negative) cells following treatment with JL189. A-B H1299 and A549 cells were treated with JL189 2.5 µM for 16 h. C Calu1 cells were treated for 2 h. The data represents the mean % change in fluorescence or mean fluorescence of 4 independent biological replicates. D-E H1299 cells treated with JL189 2.5 µM in cell culture media with and without Ca2+ for 2 h. Data represents the mean fluorescence of 3 independent biological replicates. F-G Flow cytometry analysis of the fluorescence of Rhod-2AM or Fluo-4AM in A549 WT and A549 BMPR2 KO cells. Graphs show the mean fluorescence of 4 independent biological replicates for Rhod-2AM and 3 for the Fluo-4AM studies. H Immunofluorescence imaging using a Nikon Eclipse TE300 inverted microscope of cells treated with JL189 2.5 µM then loaded with Rhod-2AM. H1299 cells were treated for 2 h and Calu-1 cells were treated for 16 h. The fluorescence of more than 25 cells was quantified by image J and is reported as the percent increase from baseline. I Calu-1 cells were treated with vehicle or JL189 2.5 µM for 16 h and loaded with Rhod-2AM and MitoTracker Green then analyzed for co-localization using a confocal microscopy. Grayscale and colored images provided. J Magnified images and the results of Menders’ and Person’s Coefficients of the co-localization analysis of Rhod-2AM and MitoTracker Green signals of all cells in the photograph (Greyscale images). K The Rhod-2AM signal was normalized to the MitoTracker Green signal in the confocal microscopy study. L H1299 cells were treated with JL189 2.5 µM for 4 h and stained with Rhod-2AM and Mitotracker Green then analyzed by flow cytometry. Graphs represent the mean value for each probe and Rhod-2AM normalized to Mitotracker Green of 3 independent biological replicates. M Calu-1 cells loaded with Rhod-2AM were placed in media without calcium then treated with JL189 2.5 µM and analyzed for 5 min by flow cytometry. Arrow marks the time of treatment. Shown is a representative study. The graph represents the mean percent increase in fluorescence from baseline of 6 independent biological replicates. N-P Calu-1 cells were loaded with Fluo-4AM, then placed in media without Ca2+ and treated with vehicle, JL189 2.5 µM (N), or Thapsigargin 300 nM (O) and analyzed for 3 min by flow cytometry. Arrows mark the time of treatment. P The graph shows the mean Fluo-4AM fluorescence of 2 independent biological replicates. Graphs show standard errors and 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