EMBO/soda-nel huggingface dataset:
New roles for Wnt and BMP signaling in neural anteroposterior patterning (2019)
Figure 1

Figure 1: Inhibition of canonical Wnt-signaling perturbs hindbrain, but not spinal cord cell fates.

A. One-cell stage embryos were injected animally with mRNA encoding dkk1 (35/45 pg) protein. Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each dkk1 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).

B. One-cell stage embryos were injected animally with the zygotic-expressing Nxfz8/pCS2 encoding plasmid (100/200 pg). Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each Nxfz8 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).

C. Embryos from the same experiment as in (A) underwent in situ hybridization. Expression of the hindbrain marker krox20 (upper two panels) was reduced by Dkk1 (45pg) in 83% of the embryos (n=18). Expression of spinal cord markers (hoxb9, cdx4, hox10) was normal, being unchanged in 89% of the embryos (n=18 for each marker). Control embryos, (CE).
New roles for Wnt and BMP signaling in neural anteroposterior patterning (2019)
Figure 2

Figure 2: Inhibition of Zic protein activity differentially affects spinal cord and hindbrain cell fates.

A. One-cell stage embryos were injected animally with the Zic1-MO (25/40 ng). Total RNA was isolated from ten control embryos at neurula stage 18 (lane 2) and ten embryos from each Zic1-MO injected group (lanes 3-4). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).

B. One-cell stage embryos were injected animally with mRNA encoding Zic5 dominant-negative (0.5 ng) protein. Total RNA was isolated from five control embryos at neurula stage 18 (lane 1) and five embryos from the injected group (lanes 2). Neural AP markers were examined by sqRT-PCR: hindbrain (krox20) and spinal cord (cdx1-4). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 3).

C. One-cell stage embryos were injected animally with the Zic1-MO (25 ng). Embryos underwent in situ hybridization. Expression of the panneural sox2 marker was normal, the forebrain marker xanf1 was expanded in 80% of the embryos, the hindbrain representative marker krox20 was reduced in 80% of the embryos and the representative spinal cord marker cdx4 was expanded in 90% of the embryos. N = 23 embryos were per marker.
New roles for Wnt and BMP signaling in neural anteroposterior patterning (2019)
Figure 3

Figure 3: BMP activity regulates neural AP patterning

A. One-cell stage embryos were injected animally with mRNAs encoding the noggin (5/20 pg) and/or meis3 (0.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 17. Total RNA was isolated from seven control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).

B. One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein. Total RNA was isolated from five control embryos at neurula stage 17 (lane 1) and five embryos from the injected group (lanes 2). Neural and mesodermal markers were examined by sqRT-PCR: forebrain (otx2), hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), panneural (nrp1, ncam) and mesodermal (muscle actin - MA). ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).

C. One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein Embryos underwent in situ hybridization. Expression of the hindbrain marker hoxb3 was reduced/eliminated by bmp4 (0.2 ng) in 68% of the embryos (n=19). Expression of the spinal cord marker cdx4 was slightly increased/normal, in 100% of the embryos (n=19).
New roles for Wnt and BMP signaling in neural anteroposterior patterning (2019)
Figure 4

Figure 4: BMP activity caudalizes ectoderm to spinal cord fates.

A. One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5-50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-7). Neural, epidermal and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10/d10, cdx1-2-4), epidermal cytokeratin (E13), lateral mesoderm (osr1-2) and dorsal-lateral mesoderm muscle actin (MA). ODC is the control for quantitating the RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).

B. One-cell stage embryos were injected animally with mRNAs encoding the bmp4 (50/150 pg) and/or meis3 (0.7 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10, cdx2), and lateral mesoderm (osr1). Ef1α is the control for quantitating the RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).

C. One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (75-200 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to gastrula stage 11-11.5. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). Marker expression was determined by sqRT-PCR. Xenopus brachyury (xbra) expression was induced by BMP4 in a dose-dependent manner. Marker expression was determined by sqRT-PCR. Neither dorsal (chd, goosecoid-gsc) or ventral-lateral (osr1) mesoderm markers were induced by BMP4. Sox2/3 neural markers are expressed in the ACs and are unchanged by BMP expression. EF1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).
New roles for Wnt and BMP signaling in neural anteroposterior patterning (2019)
Figure 5

Figure 5: BMP4 caudalizes in an FGF-dependent manner.

A. One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5-50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). FGF3-4-8 expression was determined by sqRT-PCR. ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).

B. One-cell stage embryos were injected animally with mRNAs encoding the bmp4 (50 pg) and/or FGF dominant-negative receptor (FGF-DNR, 1.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). In addition to fgf3, neural, epidermal and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), lateral mesoderm (osr1), dorsal-lateral mesoderm muscle actin (MA), and epidermal cytokeratin (E13). ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).
BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis (2021)
Figure 1

Figure 1. BNIP3 levels in melanoma patients inversely correlate with prognosis.

(A) Kaplan Meier plots representing the survival difference between SKCM patients stratified according to the 40% highest or lowest BNIP3 expression.

(B) BNIP3 levels in primary melanoma and metastases within the SKCM TCGA cohort, using Mann-Whitney's non-parametric test. The box represents the median and the 25th-75th percentile with the whiskers representing the minimum and maximum values.

(C) Representative images of BNIP3 immunostaining in a benign nevus (n=5), primary melanoma and their paired metastasis (n=7). In the representative pictures, nevi or tumor tissue is circled in yellow and highlighted with a yellow asterisk (*). Histopathological scoring of BNIP3 protein levels in nevi and melanoma patients with paired primary and metastases harboring weak and in gradient (+/-), weak and homogeneous (+) or strong and homogeneous (++) BNIP3 protein levels based on histopathological staining intensity and distribution scoring. Scale bars represent 150µm.

(D) Representative images of BNIP3 immunohistochemistry in TMAs containing (n=158) melanoma metastases and related Kaplan Meier plot of survival difference comparing melanoma patients with different BNIP3 histopathological scores. Scale bars represent 50µm.

Data information: Both Kaplan Meier curves (A, D) were analyzed using the log-rank (Mantel-Cox) test.
BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis (2021)
Figure 2

Figure 2. BNIP3 removal from melanoma cells relents tumor growth in vivo.

(A) Immunoblot detection of ATG5 and BNIP3 from lysates of CntlKD (n=5), BNIP3KD (n=5) and ATG5KD (n=7) B16-F10 tumors. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band.

(B) Growth curves of B16-F10 tumors generated with cells transduced with either empty vector (CntlKD), shRNA against BNIP3 (BNIP3KD) or ATG5 (ATG5KD) (n=7 per condition) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test.

(C) Haematoxylin and eosin staining of sections representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors. Necrotic areas are highlighted with an asterisk (*).

(D) Quantification of TUNEL+ tumor area (representing the fraction of dying/dead cell population) of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors (n= 5 per condition).

(E) Quantification of the Ki67+ cells within the tumor area (representing the fraction of proliferative cell populations in non-necrotic areas) of CntlKD (n=7), BNIP3KD (n=6) and ATG5KD (n=8) B16-F10 tumors.

(F) Immunofluorescent staining for Ki67 (red) representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumor sections.

(G) Immunofluorescent staining for LC3B (orange granularity) representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumor sections. A zoomed-in area is shown in the lower-left corner.

(H) Map1lc3b transcript levels from lysates of B16-F10 tumors [CntlKD (n=7), BNIP3KD (n=6) and ATG5KD (n=6)].

(I) Sqstm1 transcript levels from lysates of B16-F10 tumors [CntlKD (n=6), BNIP3KD (n=6) and ATG5KD (n=7)].

Data information: Scale bars represent 200µm (C) and 50µm (F, G). All quantitative data are mean ± SEM. **p < 0.01, ***p < 0.001, **** p < 0.0001 when compared against CntlKD. #p < 0.05, ##p < 0.01, ###p < 0.001 when comparing BNIP3KD against ATG5KD. Unless otherwise specified, groups were analyzed using a one-way ANOVA with Tukey's multiple comparisons test (A, D, H, I) or with Kruskal-Wallis with Dunn's multiple comparisons test (E).
BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis (2021)
Figure 3

Figure 3. BNIP3 depletion impairs mitochondrial clearance and rewires the glycolytic metabolism of melanoma cells.

(A) Confocal microscopy images of co-localization analysis of the BNIP3-proficient (shCntl, n=27 cells) or deficient (shBNIP3, n=31 cells) B16-F10 cells transiently transfected with GFP-LC3 and immunostained for TOMM20 (mitochondrial marker). Colocalization events of GFP-LC3 puncta (green) with TOMM20 (red) are highlighted with white arrows. The percentage of GFP-LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann-Whitney's non-parametric test. Scale bars represent 10µm.

(B) Schematic representation of the metabolic approaches used to characterize B16-F10 cells 1H-NMR stands for proton-based nuclear magnetic resonance, GC-MS for gas chromatography coupled to mass spectrometry, OCR for oxygen consumption rate and ECAR for extracellular acidification rate.

(C) Basal OCR per µg of protein of B16-F10 cells in the presence of galactose (25mM galactose, 2mM glutamine) medium using the Seahorse technology (n=3) analyzed using an RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test.

(D) Basal ECAR per µg of protein of B16-F10 cells assayed in glucose (25mM glucose, 2mM glutamine) medium using the Seahorse technology.

(E-G) Extracellular lactate (E), extracellular (F) and intracellular (G) glucose levels from B16-F10 cells detected using 1H-NMR (n=3) analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test.

(H) 13C incorporation in intracellular lactate from B16-F10 cells fed with 13C-labelled glucose for 24h and detected using GC-MS (n=7). The graph shows the percentage of lactate detected without any carbon isotopically labeled (m0), with one (m0+1), two (m0+2) or three (m0+3) carbons labeled. m0 and m0+3 conditions were analyzed using Friedman's test with Dunn's multiple comparisons test.

(I) Glut1, Hk2, Pkm1, Pkm2, Pdk1, Mct1, Ldha, Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16-F10 cell lysates. Transcript expression is represented as the fold-change relative to their corresponding shCntl value. Heatmap shows the average values in each condition (n=4) and they were analyzed with a one-sample T-test only against shCntl, except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test.

Data information: All quantitative data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared against shCntl. #p < 0.05, ##p < 0.01, when comparing shBNIP3 against shATG5. Unless otherwise specified, each graph represents n=4 biologically independent experiments and it was analyzed using an RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test.
BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis (2021)
Figure 4

Figure 4. BNIP3 supports normoxic HIF-1α in melanoma.

(A) Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from lysates of B16-F10 cells (n=4) or B16-F10 tumors [CntlKD (n=5), BNIP3KD (n=5) and ATG5KD (n=7)]). The HIF-1α-OH /HIF-1α ratio is shown in a bar graph below. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. Analysis was performed as indicated in the Data Information section, unless for HIF-1α/Actin ratio (in vivo), which was analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test, and LDHA/Actin ratio for shATG5 condition (in vitro), which was analyzed with Wilcoxon's rank test.

(B) Immunohistochemical staining for HIF-1α (brown) representative from CntlKD, BNIP3KD and ATG5KD, B16-F10 tumor tissues. Higher magnification of the highlighted (with a white square) stained sections is shown below.

(C, D) Glut1 (C), Pdk1 (C) and Vegfa (D) transcript levels from lysates of B16-F10 tumors [CntlKD (n=6), BNIP3KD (n=6) and ATG5KD (n=7)]. Glut1 and Vegfa were analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test.

(E-H) Double immunostaining images for CD31 and αSMA (E) and quantification of corresponding tumor vessel number (F), size (G) and maturation (H) in B16-F10 tumors (n=9 per cohort). Vessel maturation (H) was analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test.

(I) Quantification of hypoxic (pimonidazole positive) area in B16-F10 tumor tissues [CntlKD (n=9), BNIP3KD (n=9) and ATG5KD (n=8)].

Data information: All quantitative data are mean ± SEM. Scale bars (B, E) represent 50µm. *p < 0.05, **p < 0.01, ***p < 0.001 when compared against shCntl/ CntlKD. #p < 0.05, ##·p < 0.01 when comparing shBNIP3/ BNIP3KD against shATG5/ATG5KD. In vitro WB were analyzed using a one-sample T-test against shCntl whereas the hydroxylation rate was analyzed using an RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test unless otherwise stated. In vivo experiments were analyzed using a one-way ANOVA with Tukey's multiple comparisons test unless otherwise stated.
BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis (2021)
Figure 5

Figure 5. BNIP3 stabilizes normoxic HIF-1α on a post-translational level in melanoma.

(A) Immunoblot detection of PHD2 and ACTIN protein levels from lysates of normoxic cultures (n=4) or B16-F10 tumors [CntlKD (n=5), BNIP3KD (n=7) and ATG5KD (n=6)]. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. In vitro data was analyzed using a one-sample T-test against shCntl whereas the in vivo data was analyzed using a one-way ANOVA with Tukey's multiple comparisons test.

(B) Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from normoxic lysates of B16-F10 cells cultured alone or in the presence of the PHD2 inhibitor IOX2 (100µM) for 24h.

(C) Glut1, Pdk1, Mct4 and Vegfa transcript levels from B16-F10 cells cultured alone or in presence of the PHD2 inhibitor IOX2 (100µM) for 24h (n=4). Data were analyzed using a one-sample T-test against shCntl, except for Pdk1 shBNIP3 IOX2- which was analyzed using the non-parametric Wilcoxon test.

(D) Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from normoxic lysates of B16-F10 cells cultured alone or in the presence of the iron chelator DFO (200µM) for 24h.

(E) Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, PHD2 and ACTIN protein levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against PHD2.

Data information: All quantitative data are mean ± SEM. Densitometric quantifications of protein levels relative to ACTIN for (B-D-E) are shown in Appendix Table S2. *p < 0.05, **p < 0.01, ***p < 0.001 when compared against shCntl. (B, D, E) provide a representative blot from n=3 biologically independent experiments.
BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis (2021)
Figure 6

Figure 6. BNIP3 modulates intracellular iron content to support HIF-1α levels in melanoma cells.

(A) Relative intracellular Fe2+ levels in normoxic B16-F10 cells (n=4). Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. Scale bars represent 50µm.

(B) Intracellular total iron content (left) and Fe2+ to Fe3+ ratio (right) measured by CE-ICP-MS in normoxic B16-F10 cells (n=3). Data were analyzed using a one-way ANOVA with Tukey's multiple comparisons test.

(C) Immunoblot detection of NCOA4, FTL and ACTIN levels from normoxic lysates of B16-F10 cells cultured in the presence or absence of BafA 5nM for 24h (n=3).

(D) Immunoblot detection of NCOA4, PHD2 and BNIP3 levels after immunoprecipitation of endogenous BNIP3 protein from normoxic lysates of B16-F10 shCntl cells (representative of n=3 immunoprecipitations). A lysate from B16-F10 shCntl cells cultured in the presence of BafA was used for band reference. Lad. stands for the reference ladder.

(E) Intracellular Fe2+ to Fe3+ ratio measured by CE-ICP-MS in normoxic B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against NCOA4 (n=4). Data were analyzed using a one-way ANOVA with Tukey's multiple comparisons test.

(F) Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, NCOA4 and ACTIN levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against NCOA4 (n=4).

Data information: All quantitative data are mean ± SEM. Densitometric quantifications of protein levels relative to ACTIN for (C, F) are shown in Appendix Table S2. **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared against shCntl. # p < 0.05, ## p < 0.01, ### p < 0.001when comparing shBNIP3 against shATG5.
BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis (2021)
Figure 7

Figure 7: Recovery of HIF-1α levels in BNIP3-silenced cells restores melanoma glycolytic phenotype and growth in vivo.

(A) Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, Luc, LDHA, BNIP3 and ACTIN levels from normoxic lysates of shCntl and shBNIP3 cells expressing either Luc or HIF-1α-AA and collected 48h after plating (n= 4). Densitometric quantifications relative to ACTIN levels are shown below each corresponding band and they were analyzed with a one-sample T-test against shCntlLuc except for LDHA in shBNIP3 Luc, which was analyzed with Wilcoxon rank test.

(B) Glut1, Hk2, Pkm1, Pkm2, Pdk1, Ldha, Ldhb, Mct4 and Vegfa transcript levels measured by qPCR in B16-F10 cell lysates collected 48h after plating (n= 5). Transcript expression is represented as the fold-change relative to their corresponding shCntl value. Heatmap shows the average per condition and values were analyzed with a one-sample T-test against shCntl Luc except for Pdk1 in shCntl HIF-1a-AA that was analyzed with Wilcoxon rank test.

(C, D) OCR (C) and ECAR (D) per µg of protein of B16-F10 cells assayed in medium containing either 25mM galactose (C) or 25mM glucose (D) medium using the Seahorse technology (n=4).

(E) Immunoblot detection of NCOA4 and ACTIN levels from normoxic lysates of shCntl and shBNIP3 cells expressing either Luc or HIF-1α-AA, collected and treated for 24h in the absence or presence of 5nM BafA (n=3). Densitometric quantifications relative to ACTIN levels are shown below each corresponding band and they were analyzed with a one-sample T-test against shCntl Luc.

(F) Scheme explaining the feedforward loop between HIF-1α and BNIP3 in the different tumor conditions (G).

(G) Growth curves of B16-F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase (Luc) or an undegradable HIF-1α (HIF-1α-AA) construct (n=7 for CntlKD Luc/BNIP3KD HIF-1α-AA, n=8 CntlKD HIF-1α-AA and n=5 for BNIP3KD Luc) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test.

(H) Representative double immunostaining for CD31 and αSMA in B16-F10 tumors (n=5 per condition). Scale bars represent 50µm.

Data information: All quantitative data are mean ± SEM. * p < 0.05, **/$$/## p < 0.01, ***p < 0.001, ****/$$$$/#### p < 0.0001 where (*) represents comparisons against shCntl Luc/ CntlKD Luc, ($) against shCntl HIF-1α-AA/ CntlKD HIF-1α-AA and (#) against shBNIP3 Luc/ BNIP3KD Luc. Unless otherwise specified, graphs were analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test.

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