Narges Salamat

Supplementary Figure 5. The effect of HeSP2 treatment on the levels of hypoxia markers and CASP3. A, Representative IHC images of lung tissue sections from KLLuc mice showing the levels of HIF1A. B, Representative IHC images of lung tissue sections from KLLuc mice showing the levels of CA9. C, Representative IHC images of lung tissue sections from KLLuc mice showing the levels of CASP3. Shown are montage and 10X images of lung tissue sections stained with DAPI (row 1) and antibodies against the indicated protein (row 2). The light green lines in DAPI montages outline the tumors in the lung. The white rectangles denote the regions shown in 10X images. Scale bar, montage, 1mm; 10X, 100µm. D, Graph showing quantified levels of HIF1A correlating with A. E, Graph showing quantified levels of CA9 correlating with B. F, Graph showing quantified levels of CASP3 correlating with C. For statistical analysis, the levels in HeSP2-treated tumors were compared with the levels in control tumors with a Welch two-sample t test. **, P < 0.005.

Supplementary Figure 3. The levels of heme synthesis (A), heme uptake (B), and oxygen consumption rate (OCR, C) in SCC cell lines are comparable to the levels of ADC cell lines.

Supplementary Figure 1. The levels of proteins and enzyme subunits involved in OXPHOS and glutamine metabolism increase with lung tumor progression. A, Representative H&E and IHC images of lung tissue sections from KLLuc mice showing levels of the OXPHOS protein HCCS. B, Representative H&E and IHC images of lung tissue sections from KLLuc mice showing levels of the glutamine transporter SLC1A5. C, Representative H&E and IHC images of lung tissue sections from KLLuc mice showing levels of GLS. Shown are montage images of lung tissue sections stained with H&E (row 1), montage images stained with DAPI (row 2) or antibodies against the indicated protein (row 3), and 10X images of lung tissue sections stained with DAPI (row 4) or antibodies against the indicated protein (row 5). The light green lines in DAPI montage images outline the tumors in the lung. The blue rectangles in H&E and white rectangles in DAPI montage images denote the regions shown in 10X. Scale bar, montage, 1mm; 4X, 200 µm; 10X, 100µm. D, Graph showing quantified levels of HCCS correlating with A. E, Graph showing quantified levels of SLC1A5 correlating with B. F, Graph showing quantified levels of GLS correlating with C. Protein levels were quantified, and data are plotted as mean {plus minus} SEM. G, Representative 40X IHC images of lung tissue sections showing the co-localization of ATP5F1A, COX7B, and CYCS with the mitochondrial marker TOMM20. Scale bar, 10 µm. H, OCR of NSCLC H226 cells cultured in normal medium, or heme depleted medium with 0.5 mM succinyl acetone (HD+SA). Heme (5 µM, 10 µM, and 20 µM, respectively) was added back to examine the effects of heme on OCRs. For statistical analysis, the levels in cells with heme added back were compared with the levels in cells without heme added back with a Welch two-sample t test. **, P<0.005.

Supplementary Figure 2. The levels of representative OXPHOS complex subunits in ADC and SCC tumors developed in KLLuc mice. A, Representative IHC images of lung tissue sections showing levels of COX5B, along with ADC marker NKX2-1 and SCC marker Î"Np63, from KLLuc mice at 10 weeks after the initiation of tumorigenesis via viral infection. B, Representative IHC images of lung tissue sections showing levels of COX7B, along with ADC marker NKX2-1 and SCC marker Î"Np63, from KLLuc mice at 10 weeks after the initiation of tumorigenesis via viral infection. C, Representative IHC images of lung tissue sections showing levels of CYCS, along with ADC marker NKX2-1 and SCC marker Î"Np63, from KLLuc mice at 10 weeks after the initiation of tumorigenesis via viral infection. Shown are montage and 10X images of lung tissue sections stained with DAPI (row 1), antibodies to NKX2-1 or Î"Np63 (row 2), antibodies to the indicated protein (row 3), and merged images of NKX2-1 or Î"Np63 with the indicated protein (row 4). The light green lines in DAPI montage images outline the tumors in the lung. The white arrows denote the regions shown in 10X images. Scale bar, montage, 1mm; 10X, 100µm. D, Graph showing quantified levels of COX5B in ADC and SCC tumors correlating with A. E, Graph showing quantified levels of COX7B in ADC and SCC tumors correlating with B. F, Graph showing quantified levels of CYCS in ADC and SCC tumors correlating with C. Protein levels were quantified, and data are plotted as mean {plus minus} SEM.

Figure S6. HeSPs strongly reduce the levels of heme transporters without affecting iron metabolism in subcutaneous NSCLC tumor xenografts. A, Representative IHC images of H1299 NSCLC tumor tissue sections and graphs showing the levels of SLC48A1 in control and HeSP-treated tumors. B, Representative IHC images of H1299 NSCLC tumor tissue sections and graphs showing the levels of SLC46A1 in control and HeSP-treated tumors. C, Representative IHC images of H1299 NSCLC tumor tissue sections and graphs showing the levels of FLVCR2 in control and HeSP-treated tumors. D, Representative IHC images of H1299 NSCLC tumor tissue sections and graphs showing the levels of ferroportin 1, SLC40A1, in control and HeSP-treated tumors. E, Representative IHC images of H1299 NSCLC tumor tissue sections and graphs showing the levels of transferrin receptor TFRC in control and HeSP-treated tumors. Shown IHC figures are montages or 10X images of control and HeSP-treated tumor tissue sections stained with DAPI or antibodies against the indicated protein. The white rectangles in DAPI images denote the regions shown in 10X images. Liver is shown as a positive control for the detection of all proteins shown. Scale bar: montage, 1 mm; 10X, 200 μm. F, Representative 40X IHC images of H1299 NSCLC tumor tissue sections showing the colocalization of FLVCR2, SLC46A1, TFRC, and SLC40A1 with membrane marker PMCA1. Scale bar, 20 µm. G, Iron stain did not detect noticeable difference between control and treated tumor tissues. (Left) Shown are montages, 10X, and 40X images of control and HeSP-treated tumor tissue sections stained with iron stain. Liver is shown as a positive control for iron stain. The black rectangles in montages, 10X images are the regions shown in 10X and 40X images, respectively. Scale bar: montage, 1 mm; 10X, 200 µm; 40X, 50 µm. (Right) The levels of iron in tumor lysates from control mice and mice treated with HeSPs. Liver lysates were used as a positive control while BSA solution was used as a negative control. Protein and iron levels were quantified. Data were plotted as mean {plus minus} SEM. **, P < 0.01; ***, P < 0.005.