EMBO/SourceData huggingface dataset:
Figure 5In vitro cleavage assay of SARS-CoV-2 spike protein. Recombinant spike protein (1 µg) was digested with ADAM10, TMPRSS2 (A) for 24 hours at a 1:1 ratio and analyzed by Western Blot. Camostat mesylate or BB94 (10 µM) were pre-incubated with TMPRSS2 or ADAM proteases, respectively, for 15 min before addition of spike protein. The spike protein and fragments were detected using an antibody against its C-terminal 6xHIS tag.B. In vitro cleavage assay of SARS-CoV-2 spike protein. Recombinant spike protein (1 µg) was digested with ADAM17 (B) for 24 hours at a 1:1 ratio and analyzed by Western Blot. BB94 (10 µM) were pre-incubated with ADAM proteases, respectively, for 15 min before addition of spike protein. The spike protein and fragments were detected using an antibody against its C-terminal 6xHIS tag.C. Representative images and quantification of a proximity ligation assay (PLA) between the SARS-CoV-2 Spike and ADAM17 in A549-ACE2 cells. Cells were infected with SARS-CoV-2 and placed on ice (4°C). After 2 h, cells were shifted to 37°C for 15 min or left on ice. Images are presented as overlay between the Nuclei (Hoechst, blue) and the PLA (pseudo-color: red) channel. Scale bar, 20 µm. The number of PLA foci per cell was determined for the acquired images (N = 28-30). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.D. A549-ACE2 cells were treated for 48 h with BB94 (10 µM), DPC-333 (5 µM) or DMSO. PMA (25 ng/ml) was added for 3 h before harvest to stimulate ACE2 shedding. Concentration of the soluble ACE2 fragment was determined in the conditioned medium by ELISA and is shown relative to the concentration in the DMSO control (N = 6). One-way ANOVA with Tukey's post-hoc multiple comparison test. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.E. GFP-encoding spike pseudoparticles (VSV pseudotyped with SARS-CoV-2 spike protein (VSV-S) were incubated with indicated amounts of recombinant, soluble ACE2 (sACE2) or bovine serum albumin (BSA) as a control prior to infection of A549-ACE2 cells. At 16 hpi, infection was analyzed by counting GFP-positive cells. Data are normalized to the number of infected cells in virus-only control wells without sACE2 or BSA (N = 3). Two-sided independent Student's t test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
f1(a) Expression of Ulk1 and Atg5-12 in the indicated erythroid cells.(b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA).(b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA).
f2(a) Induction of alternative macroautophagy in WT and Atg5−/−, but not Ulk1gt/gt and DKO, erythroid cells. Erythroblasts and reticulocytes from the liver of embryonic mice (E18.5) were incubated with or without STS (1 μM) for 24 h, followed by staining with anti-Lamp2 (red), anti-Ter119 (green) and DAPI (blue). DAPI-positive erythroblasts and DAPI-negative reticulocytes are shown. Lamp2 image and merged image (DAPI, Ter119 and Lamp2) are shown. Scale bar, 1 μm. Large dots for Lamp2 are observed in STS-treated WT and Atg5−/− cells, but not STS-treated Ulk1gt/gt or DKO cells.(b) Representative electron micrographs of EC incubated with or without STS. Erythroid cells were harvested from the liver of embryonic mice (E18.5), incubated with or without STS (1 μM) for 24 h, and analysed by electron microscopy (EM). Scale bar, 1 μm. Insets of WT and Atg5−/− cells show mitophagy (Scale bar, 0.5 μm). Inset of a Ulk1gt/gt cell showing mitochondria that have not been engulfed (Scale bar, 0.5 μm). Arrows point to non-engulfed mitochondria and the arrowheads indicate engulfed mitochondria. (c-e) Quantitative analysis of mitophagy after STS treatment, calculated from EM photos. Population of reticulocytes with mitophagy (c), number of mitochondria per reticulocytes (d) and population of reticulocytes showing macroautophagy (e) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).
f3(a) Three-colour flow cytometry of erythroid cells from WT embryonic liver (E14.5) stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red. (Left upper panel) Ter119 versus CD71 fluorescence. (Other panels) FSC versus Mitotracker fluorescence of the R1 (right upper panel), R2 (left lower panel) and R3 (right lower panel) fractions. (b,c) Percentage of cells with a low number of mitochondria (mitochondrialow cells) among CD71−Ter119+ EC. Erythroid cells from the liver (E14.5 and E18.5) and peripheral blood (E18.5) of indicated mice were stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red.(b) Representative histograms of mitochondrial content in CD71−Ter119+ cells. Numbers indicate the population of mitochondrialow cells.(c) The percentage of CD71−Ter119+ cells without mitochondria was determined by gating the mitochondrialow fraction, as shown in (b) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).(d) Percent mitochondrialow cells in the CD71−Ter119+ EC of Atg7F/FCre embryo. Erythroid cells from the liver and peripheral blood of WT and Atg7F/FCre embryo (E18.5) were stained with anti-Ter119, anti-CD71, and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test).(e) Percentage of mitochondrialow cells in Syto16low cells. Liver erythroid cells (E18.5) were stained with Syto16 (DNA) and Mitotracker Deep Red. Representative dot plots of the mitochondrial content of Syto16low cells are demonstrated in Supplementary Fig. 5, the percentage of Syto16low cells without mitochondria was determined by gating the mitochondrialow fraction (mean±s.d., n=6). *P0.05 versus value of WT (ANOVA); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).(f) Immunofluorescent analysis of mitochondrial and lysosomal proteins. Liver Ter119+ cells (E18.5) of the indicated mice were stained with anti-Tom20 (mitochondrial marker) and anti-Lamp2 (lysosomal marker) antibodies, and observed by fluorescent microscopy. Scale bar, 2 μm. Green, red, white, and blue indicate Tom20, Lamp2, Ter119 and DAPI (DNA), respectively. Mitochondrial and lysosomal markers have separate distributions in Ulk1gt/gt cells and DKO cells.
f4(a-f) Representative EM of WT (a), ATG5−/− (b), Ulk1gt/gt (c,d) and DKO (e,f) reticulocytes from embryonic liver (E14.5). (a,b) Mitophagy was detected in WT and ATG5−/− cells (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm). (c,e) In Ulk1gt/gt and DKO cells, numerous mitochondria were in contact with membrane structures (scale bar, 1 μm). Enlarged images are shown on the right side (scale bar, 0.2 μm). (d,f) Some Ulk1gt/gt and DKO cells showed plasma membrane blebs containing mitochondria (scale bar, 1 μm). (g,h) Quantitative analysis of mitophagy calculated from EM photos in the embryonic liver at E18.5. Population of reticulocytes with mitophagy (g), and number of mitochondria per reticulocytes (h) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).
f5(a) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated embryos (E18.5), and the expression of each protein was examined by western blot. The asterisk indicates a nonspecific band. Uncropped images are shown in Supplementary Figs 11 and 12.(b) Semi-quantitative analysis of the expression level of each protein and the LC3-II/LC3-I ratio (mean±s.d., n=3). *P0.05 versus value of WT EC (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− EC (ANOVA); 'NS' indicates not significant versus value of WT EC (ANOVA).(c) Modification of Ulk1 and Atg13 during erythrocyte maturation. Cell lysates were treated with λ-phosphatase (400 U) for 45 min at 30 °C, and band shift was examined by anti-Ulk1 and anti-Atg13 antibodies. Asterisk indicates non-specific band. Uncropped images are shown in Supplementary Fig. 12.
f6(a) Morphology of cultured erythroid cells on day 0, 1 and 3 of differentiation. Erythroblasts were purified from embryonic liver (E14.5) and incubated for the indicated times in erythrocyte differentiation medium. The cells were stained with a Diff-quick kit. (b) Percentage of cells without nuclei (Syto16low cells) on the indicated days of differentiation (mean±s.d., n=6). Loss of nuclei occurred normally in all mice.(c,d) Percentage of Syto16low cells without mitochondria. Differentiated cells were stained with Syto16 and Mitotracker Deep Red. Representative histograms of mitochondrial content in Syto16low cells on day 6 are shown in (c). Numbers indicate the population of mitochondrialow cells. Representative dot plots are also available in Supplementary Fig. 13. (d) Percentage of Syto16low cells lacking mitochondria determined by gating the mitochondrialow fraction as shown in (c) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).(e-h) Impact of various drugs on mitochondrial clearance during in vitro differentiation. Differentiated cells from WT embryos were incubated with or without wortmannin (e), 3-methyladenine (3-MA) (f), rapamycin (g) or compound C (h) at the indicated concentrations and time periods. Percentage of mitochondrialow cells/Syto16low cells was measured by flow cytometry (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).(i)Impact of various drugs on mitochondrial clearance during in vitro differentiation.with cells from Atg5−/− embryos exposed to wortmannin or 3-MA (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).
f7(a) Number of EC and haemoglobin content in the blood of the indicated mice (10-20 weeks old; mean±s.d., n=6). *P0.05 versus value of WT (Student's t-test).(b) Analysis of early erythrocyte development. Bone marrow cells were harvested from the indicated mice (10-20 weeks old) and stained with anti-cKit and anti-Sca1 antibodies. The population of each cell type was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)).(c) Percentage of mitochondria-containing Ter119+ blood cells. Cells in peripheral blood from the indicated mice (10-20 weeks old) were stained with anti-Ter119 antibody together with Mitotracker Deep Red. The population of mitochondriahigh cells among Ter119-positive cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA).(d) Percentage of mitochondria-containing Syto16low blood cells. Cells in peripheral blood from the indicated mice (10-20 weeks old) were stained with Syto16 and Mitotracker Deep Red. The population of mitochondriahigh cells among Syto16-negative cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA).(e) Representative electron micrographs of bone marrow reticulocytes in 10 week-old WT and Ulk1gt/gt mice (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm).(f,g) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated mice (10-20 weeks old), and the expression of each protein was examined by western blot. Uncropped images are shown in Supplementary Fig. 14. (g) Semi-quantitative analysis of the expression level of each protein (mean±s.d., n=3). 'NS' indicates not significant versus value of WT EC (ANOVA).(h,i) The impact of Ulk1 on mitochondrial clearance during stress erythropoiesis. (h) Representative images of erythrocyte in PHZ-treated WT and Ulk1gt/gt mice (14 weeks old). Blood cells were stained with anti-Ter119 (erythroid cell marker) and anti-Tom20 (mitochondrial marker) antibodies and observed by fluorescence microscopy. Green and red indicate Ter119 and Tom20, respectively. Scale bar, 10 μm. (i) Percentage of mitochondrialow cells among Syto16low cells. Erythroid cells were stained with Syto16 (DNA) and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test). Mitochondrial retention was observed in Ulk1gt/gt mice.
Figure 3A. STAT3 immunohistochemistry- staining of low STAT3 and high STAT3 PCa samples. Red arrows indicate transformed PCa glands, black arrows indicate pre-transformed normal prostate glands. Scale bar = 100µm. # = sample-IDs.
Figure 4A. Differentially expressed proteins in PtenStat3pc-/- vs. Ptenpc-/- proteomic samples. Colors indicate adj.p-value and log2-FC. (Black = Log2-FC ≤ 1 & adj. p-value ≥ 0.05, orange = Log2-FC > 1 & adj. p-value ≥ 0.05, red = Log2-FC > 1 & adj. p-value < 0.05). FC = fold change.B. STAT3 immunohistochemistry- staining of wild type, Ptenpc-/-, PtenStat3pc-/- mouse prostates. Scale bar = 100µm.E. Metabolite concentrations in nmol/µg of 5 metabolites in WT, Ptenpc-/- and PtenStat3pc-/- prostates. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents biological replicates. ANOVA tests and Tukey multiple comparisons were applied. Red = PtenStat3pc-/- (n = 3), blue = Ptenpc-/- (n = 5), grey = wild type (WT, n=5), q = adj. p.-value, n.s. = not significant.
Figure 5A. SDHB protein expression levels in a TMA (n = 83) detected by immunohistochemistry (IHC). Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant. TMA = Tissue Micro Array.B. Representative IHC- staining of SDHB in normal prostate glands and Gleason grade (GL) 3 - 5 PCa glands. Scale bar = 100µm.C. IDH2 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.D. Representative IHC staining of IDH2 in normal prostate glands and GL 3 - 5 PCa glands. Scale bar = 100µm.E. Nuclear (N) and cytoplasmatic (C) STAT3 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.
Figure 6B. Genes of the Hallmark gene set "Oxidative phosphorylation" that are differentially expressed in low STAT3 vs. high STAT3 tumors. Dotted lines show Log2-FC = ±1 (x-axis) and adj. p-value = -log10(0.05) (y-axis). Orange = up-regulated genes, green = down-regulated genes. DE = differentially expressed.C.- D. Kaplan-Meier plots showing time to biochemical recurrence in months for PDK4 in primary tumors (C) and in primary and metastatic tumors combined (D) in the MSKCC PCa GSE21032 data set. Groups were generated by a median split. P-values were estimated by Log-rank test (C-D) and adjusted with Benjamini-Hochberg method (D). + = censored.
Figure 7A. STAT3 and PDK4 stratified subgroups were generated by median splits in MSKCC PCa GSE21032 data set. Pearson correlation between STAT3 and PDK4 is shown. Kaplan-Meier plot shows stratified subgroups. P-values were estimated by Log-rank test and adjusted with Benjamini-Hochberg method. Hi = high, lo = low.B. Western blot of STAT3, PDK4 and β-TUBULIN proteins in 22Rv1 cells with or without knockdown of STAT3. Ctrl = scrambled control, shSTAT3 = short hairpin knockdown of STAT3.C. ChIP assay from IL-6 stimulated or non-stimulated 22Rv1 cells with or without knockdown of STAT3 was immunoprecipitated with a STAT3 specific antibody (blue shades) and IgG antibody as negative control (orange shades) followed by qPCR with a promoter- specific primer pair for the PDK4 gene. Bars represent mean ± SD from 2 technical replicates. Precipitated DNA is presented as % of input. One representative experiment is shown. Result of qPCR using primer pair 2 is shown. Ctrl = scrambled control, shSTAT3 = short hairpin knockdown of STAT3, +IL-6 = IL-6 stimulated.D. Correlation of STAT3, c-MYC and HIF-1α with PDK1-4, PDC- genes (PDHA1, PDHB, PDHX, DLAT, DLD) and TCA/OXPHOS genes (CS, IDH2, IDH3A, SDHB, SDHC, ATP5A1, NDUFS1) in MSKCC PCa (GSE21032). Dot colors represent Pearson correlation (1 = red, -1 = blue), dot sizes represent adj. p-values ≤ 0.05. Only significant correlations are shown. P-values were adjusted with Benjamini-Hochberg method.
Figure 1(A) BMDMs were treated with E. coli (MOI=100) for 30 minutes and further cultured for various length of time as indicated. The dynamic of ENT3 expression was determined by measuring Slc29a3 transcripts via RT-qPCR. (B) Phagocytosis inhibitor, Cytochalasin D (CytoD), pretreated BMDMs were incubated with E. coli (MOI=100) and subjected for Slc29a3 expression analysis via RT-qPCR 6 hours post-infection. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(D) Different bacterial components were applied as stimulants to BMDMs. Slc29a3 expression was analyzed after 30 minutes of stimulation and a total of 6 hours incubation. (E) BMDMs were treated with different doses of LPS for 30 minutes, harvested 6 hours post-stimulation, and subjected for RT-qPCR analysis. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.
Figure 2(C) WT or (D) MyD88-/- BMDMs were stimulated with 10 ng/ml LPS or live E. coli (MOI=100) in the presence of 10 µM TBK inhibitor, MRT 67307, and analyzed for Slc29a3 expression. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(E) The 10 ng/ml LPS-treated BMDMs were examined for Ifnb1 expression after 30 minutes of stimulation and a total of 6 hours incubation. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(F) BMDMs were treated with 100 U/ml of IFN-β for various length of time and subjected for Slc29a3 expression. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.
Figure 3(B-E) BMDMs from WT, IFNAR1-/- (B), or STAT1-/- (C) mice were treated with 10 ng/ml LPS, live E. coli (MOI=100), or 100 U/ml IFN-β, harvested and examined for Slc29a3 induction. BMDMs from WT, IFNAR1-/- (D) or STAT1-/- (E) mice were treated with 100 U/ml IFN-β, 50 µg/ml poly(I:C), or EMCV (MOI=10), harvested and examined for Slc29a3 induction. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown in B to E are from three biological replicates (n=3) Unpaired two-tailed Student's t-test was used for statistical analysis, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(F-H) The expression dynamic of Slc29a3 was profiled in BMDMs treated with two different doses of IFN-α (F), IFN-β (G), IFN-γ (H) for various length of time as indicated. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown in F to H from two biological replicates (n=2). Unpaired two-tailed Student's t-test was used for statistical analysis, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.
Figure 4(A) The induction fold change (treated/untreated) of Slc29a3 and selected ISG transcripts were curated from INTERFEROME database. Shown is data extracted from 250 IU/ml IFN-β treated BMDMs. Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(D-F) Primers targeting 3' flanking region 2 (D), 3' flanking region 1 (E), and promoter region (F) were used to quantify the STAT1 binding in BMDMs upon 100 U/ml IFN-β after 4 hour stimulation via qChIP assay. Data information: The results in D-F were generated from two biological replicates (n=2) with four technical repeats each (n=4). One representative example is shown. Unpaired two-tailed Student's t-test was used for statistical analysis, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.