Journal: EMBO Molecular Medicine

Article Title: Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities

doi: 10.1038/s44321-025-00361-w

Figure Lengend Snippet: ( A ) VHL dependency score in ccRCC cell lines ( N = 21) and other pan cancer lines ( N = 1162). Unpaired Wilcoxon test to calculate significance. The horizontal line in the box marks the median (Q2), the upper and lower hinges correspond to the 25th (Q1) and 75th (Q3) percentiles. The whiskers extend to 1.5× the interquartile range from Q1 to Q3. ( B ) Quantification of normal renal organoid growth (EMKC016) with and without VHL inactivation, N = 32 random growing organoids per condition and time point (mean and S.E.M.). ( C ) Activity of a hypoxia reporter (HRE-ODD-GFP) in normal renal organoids (EMKC016) with and without VHL deletion as determined by fluorescence microscopy. ( D ) Schematic of the pooled CRISPR-Cas9 screening strategy. ( E ) Gene level CRISPR-Cas9-based loss of function screening data. Beta scores showing change in sgRNA construct abundance in VHL mutant cells. N = 2 replicates per condition. ( F ) CRISPR-Cas9 based competition assay in VHL mutant MUT10 cells. HIF1A, HIF2A and ARNT mutant cells competed against cells transduced with non-targeting control constructs (NTC). Two sgRNAs per gene combined, N = 3 technical replicates per condition (mean and S.E.M.). ( G ) VHL mutant human renal epithelial organoids with or without HIF1A inactivation at different time points. ( H ) Quantification of organoid growth from ( G ) over time, N = 13 random growing organoids per condition and time point (mean and S.E.M.). .

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore) and blotted with VHL (BD Pharmingen, 565183, 1:500), HIF1A (Proteintech, 20960-1-AP, 1:1000), HIF2A (Novus Biologicals, NB100-122, 1:1000), beta-actin (Sigma-Aldrich, A1978, 1:30,000), p-ERK (Abcam, ab201015, 1:1000), ERK (Abcam, ab184699, 1:2000) antibodies.

Techniques: Activity Assay, Fluorescence, Microscopy, CRISPR, Construct, Mutagenesis, Competitive Binding Assay, Transduction, Control

Journal: EMBO Molecular Medicine

Article Title: Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities

doi: 10.1038/s44321-025-00361-w

Figure Lengend Snippet: ( A ) Proliferation of HK2 cells with or without VHL. N = 2 per condition. (mean and S.E.M.). ( B ) Western blot of HIF2A, VHL and Actin on HK2 cells with and without VHL. ( C ) Morphology of HK2 cells with and without VHL. ( D ) Proliferation of VHL mutant HK2 cells with and without VHL re-introduction. N = 3 per condition (mean and S.E.M.). ( E ) Western blot of HIF2A on VHL mutant HK2 cells with and without VHL re-introduction. ( F ) Proliferation of HK2 cells with and without VHL under 5% or 21% O 2 culture conditions. N = 2 per condition (mean and S.E.M.). ( G ) Representative images of human renal epithelial organoids with and without VHL. N = 4 replicates per condition. ( H ) Human renal epithelial organoid proliferation under DMSO or DMOG (3 mM) treatment.

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore) and blotted with VHL (BD Pharmingen, 565183, 1:500), HIF1A (Proteintech, 20960-1-AP, 1:1000), HIF2A (Novus Biologicals, NB100-122, 1:1000), beta-actin (Sigma-Aldrich, A1978, 1:30,000), p-ERK (Abcam, ab201015, 1:1000), ERK (Abcam, ab184699, 1:2000) antibodies.

Techniques: Western Blot, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities

doi: 10.1038/s44321-025-00361-w

Figure Lengend Snippet: ( A ) Schematic of doxycycline (dox) inducible VHL re-introduction into VHL mutant (MUT10 and MUT35) and wild-type control (WT8) clones. ( B – D ) Proliferation of WT8 ( B ), MUT10 ( C ) and MUT35 ( D ) cells with and without dox. N = 2 replicates per condition (mean and S.E.M.). ( E ) Western blot of HIF1A, HIF2A, VHL and Actin on WT8, MUT10 and MUT35 cells with and without dox. ( F ) Cas9 editing efficiency tested on WT8, MUT10 and MUT35 cells by a reporter plasmid using fluorescence-activated cell sorting.

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore) and blotted with VHL (BD Pharmingen, 565183, 1:500), HIF1A (Proteintech, 20960-1-AP, 1:1000), HIF2A (Novus Biologicals, NB100-122, 1:1000), beta-actin (Sigma-Aldrich, A1978, 1:30,000), p-ERK (Abcam, ab201015, 1:1000), ERK (Abcam, ab184699, 1:2000) antibodies.

Techniques: Mutagenesis, Control, Clone Assay, Western Blot, Plasmid Preparation, Fluorescence, FACS

Journal: EMBO Molecular Medicine

Article Title: Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities

doi: 10.1038/s44321-025-00361-w

Figure Lengend Snippet: ( A ) CRISPR/Cas9-based genome wide screen data. sgRNA abundance on day 28 relative to start of the assay in VHL mutant clones MUT10 and MUT35. R, Pearson’s correlation coefficient. ( B , C ) As in ( A ) with sgRNAs targeting genes of interest highlighted in red: HIF1A in ( B ) and ARNT in ( C ). ( D ) Pathway enrichment analysis on the top 500 genes the sgRNAs of which are depleted over time in MUT10 and MUT35 cells using the Cancer Hallmarks gene sets. ( E ) A schematic of the competitive proliferation assay. VHL-HIF1A, VHL-HIF2A and VHL-ARNT double mutant cells (BFP labelled) competed against VHL-NTC single mutant cells (GFP labelled). ( F – H ) Western blot of HIF1A, ARNT and HIF2A on MUT10 cells with and without HIF1A, ARNT or HIF2A inactivation, respectively. ( I ) Morphology of VHL-NTC, VHL-HIF1A, VHL-HIF2A and VHL-ARNT cells.

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore) and blotted with VHL (BD Pharmingen, 565183, 1:500), HIF1A (Proteintech, 20960-1-AP, 1:1000), HIF2A (Novus Biologicals, NB100-122, 1:1000), beta-actin (Sigma-Aldrich, A1978, 1:30,000), p-ERK (Abcam, ab201015, 1:1000), ERK (Abcam, ab184699, 1:2000) antibodies.

Techniques: CRISPR, Genome Wide, Mutagenesis, Clone Assay, Proliferation Assay, Western Blot

Journal: JCI Insight

Article Title: Identification of HIF1A as a therapeutic target during SARS-CoV-2–associated lung injury

doi: 10.1172/jci.insight.191463

Figure Lengend Snippet: ( A ) Schematic diagram of vadadustat treatment in HIF reporter ODD-luc mice. ( B ) Representative ex vivo bioluminescence imaging of luciferase activity using IVIS imager at 2 hours after vadadustat i.p. injection. ( C ) Quantification of bioluminescence intensity at 2 hours and 4 hours after vadadustat i.p. injection. Data are presented as mean ± SD. ( D ) Schematic diagram of vadadustat treatment in C57BL/6 mice. ( E ) Hif1A or Hif2A immunoblotting was performed on protein isolated from whole lung tissue after treatment with vadadustat. Each column represents 1 animal. ( F and G ) Quantification of Hif1a and Hif2a protein after treatment with vadadustat for 3 days. Data are presented as mean ± SD. ( H ) Schematic diagram of WA1 infection (280 PFU) in K18-hACE2 mice treated with vadadustat. ( I ) Kaplan-Meier plots of K18-hACE2 mice with vehicle or vadadustat treatment. P values were calculated with the Mantel-Cox test. ( J ) Blinded histological injury scores of the lungs were quantified as described in the Methods. Data are represented as mean ± SEM. ( K ) Representative H&E staining images of lung tissue from vehicle- and vadadustat-treated mice. Scale bars: 50 μm. ( L ) Schematic diagram of MA10 infection (200 PFU) in BALB/c mice treated with vadadustat. ( M ) Kaplan-Meier plots of BALB/c mice with vehicle or vadadustat treatment. P values were calculated with the Mantel-Cox test. * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( C ) or 2-tailed Student’s t test ( F , G , and J ).

Article Snippet: Membranes were probed with respective primary antibodies targeting Hif2a (NB 100-122, Novus; diluted 1:2000 in PBST), Hif1a (14179, Cell Signaling Technology; diluted 1:2000 in PBST), or α-tubulin (2144, Cell Signaling Technology; diluted 1:2000 in PBST) and incubated overnight at 4°C with gentle swirling.

Techniques: Ex Vivo, Imaging, Luciferase, Activity Assay, Injection, Western Blot, Isolation, Infection, Staining, Comparison

Journal: JCI Insight

Article Title: Identification of HIF1A as a therapeutic target during SARS-CoV-2–associated lung injury

doi: 10.1172/jci.insight.191463

Figure Lengend Snippet: ( A ) Mice were inoculated with 3 × 10 4 PFU of the murine-adapted SARS-CoV-2 strain (MA10) via oropharyngeal aspiration, and clinical outcomes were monitored over 7 days. ( B and C ) The survival rate in SARS-CoV-2–infected mice with whole-body deletion of Hif1a ( Hif1a fl/fl UBCCreER) or ( C ) Hif2a-deleted mice ( Hif2a fl/fl UBCCreER) compared to their respective Hif fl/fl litter mates. P values were obtained using the Mantel-Cox test. ( D ) Mice with a specific deletion of Hif1a in alveolar epithelial cells ( Hif1a fl/fl SPCCreER) and their Cre-inducible counterpart (SPCCreER) were infected with 3 × 10 3 PFU of the MA10 strain via oropharyngeal aspiration or mock infected, monitored for clinical outcomes and euthanized on day 4 to harvest BALF and lung tissue. ( E ) Albumin concentration in BALF was measured by ELISA. Data are represented as mean ± SEM. Two-tailed Student’s t test. ( F and G ) Viral load in BALF and lung tissue was detected by plaque assay. Gaussian distribution was assayed using the Shapiro-Wilk test. Unpaired 2-tailed Student’s t test or Mann-Whitney U test was applied to parametric or nonparametric data, respectively. ( H ) The lungs of infected SPCCreER and Hif1a fl/fl SPCCreER mice 4 days after infection were collected, fixed, and paraffin embedded. H&E staining was performed, and images were taken at ×10 magnification ( n = 5 or 8, respectively; representative images are shown). Scale bars: 200 μm. ( I ) The lung injury score was performed blindly. In the bar-and-whisker plots, the bounds of the boxes represent the 25%–75% interquartile range, the lines within the boxes represent the median, the whiskers represent data min/max, and there are no outlying values. Two-tailed Student’s t test. ( J ) Inflammatory molecules were measured using a multiplex array in the BALF from SPCCreER and Hif1a fl/fl SPCCreER SARS-CoV-2– or mock-infected mice. Volcano plot resulting from an unpaired 2-tailed Student’s t test with Welch’s correction comparing both groups. Molecules that were highly differentially secreted are emphasized in red. The column graphs represent individual results for IL-6, G-CSF, and IP-10 ( n = 10–12). Unpaired 2-tailed Student’s t tests with Welch’s correction or Mann-Whitney U test was applied to parametric or nonparametric data. Normality was established using the Shapiro-Wilk test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Membranes were probed with respective primary antibodies targeting Hif2a (NB 100-122, Novus; diluted 1:2000 in PBST), Hif1a (14179, Cell Signaling Technology; diluted 1:2000 in PBST), or α-tubulin (2144, Cell Signaling Technology; diluted 1:2000 in PBST) and incubated overnight at 4°C with gentle swirling.

Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Plaque Assay, MANN-WHITNEY, Staining, Whisker Assay, Multiplex Assay