Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Expression qPCR analysis for HIF-2α mRNA in tissue lysates from renal tumors or corresponding normal kidney tissue from the Erlangen RCC cohort (ccRCC: clear cell renal carcinoma [ n = 114], pRCC: papillary renal cell carcinoma [ n = 16], chRCC: chromophobe renal cell carcinoma [ n = 11]). Data shown are median and interquartile range. Significance was tested by two-way ANOVA followed by Bonferroni’s post hoc test. b RNA-seq expression values for HIF-2α in different renal tumors and corresponding normal kidney tissue from the TCGA KIRC ( n = 72), KIRP ( n = 31) and KICH ( n = 23) data sets. Median and interquartile range. P -values were determined by two-way ANOVA followed by Bonferroni’s post hoc test. c RNAscope experiment for HIF-2α mRNA in ccRCC tissue. Arrows indicate HIF-2α mRNA transcripts in tumor cells. V = vessel. Scale bars, 100 µm or 75 µm as indicated. Representative image from independent samples of seven different patients with similar results. d Log 2 fold change of HIF-related genes in snRNA-seq data comparing tumor cells from 30 ccRCC samples versus proximal tubule (PT) cells from four adjacent kidney tissue samples . Only samples with significantly differentially expressed transcripts are shown (adjusted p < 0.05, average log 2 fold change ≠ 0). e Isolation of renal tumor cells and corresponding tubule cells from non-diseased tissue of tumor nephrectomy specimens. Created in BioRender. Naas, S. (2025) https://BioRender.com/c87s173 . f Immunoblot analyses for HIF-1α, HIF-2α, pVHL, and β-actin in lysates from primary tubule (PTC) and tumor (ccRCC) cells isolated from tissue specimens of patient #1. Representative blot from 19 independent experiments with similar results. g Expression qPCR analysis for HIF-2α mRNA in lysates from primary tubule cells (PTC) and primary tumor (ccRCC) isolated from 34 different individuals. Graph shows median with interquartile range and p -values from a two-tailed unpaired t-test.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Expressing, RNA Sequencing, RNAscope, Isolation, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Sequencing tracks from ATAC- and H3K27ac ChIP-seq experiments performed in cells from patient #1 at the EPAS1 locus. Activity-by-contact (ABC) analysis was used to predict enhancer-promoter interactions. Additionally, tracks from published data from Capture-C experiments in 786-0 cells , gained enhancer activity in ccRCC (ccRCC-enhancer) , ccRCC cancer-cell specific differentially accessible regions (ccRCC-specific DACR) as determined by snATAC-seq , KIRC hypomethylated CpGs (Beta-value cutoff 0.25, adjusted p -value cutoff 0.01) and KIRC-specific ATAC-seq elements are shown . b Differentially active enhancer regions on chromosome 2 in ccRCC tumors from Yao et al . Average log 2 fold chance (H3K27ac signal, ten tumors vs corresponding normal tissue) and -log 10 adjusted p -value are shown. Significantly regulated elements (adjusted p < 0.05, light blue) that overlap KIRC-specific ATAC sites are depicted in dark blue. Sites highlighted in red overlap KIRC-specific sites, show a positive log 2 fold change and are localized within 5 Mb of the EPAS1 gene. Enhancer enh-1031503 covers elements KIRC_10767-KIRC_10770. For comparison, the H3K27ac signal at the EPAS1 promoter is marked, but not significantly regulated. c ATAC-seq tracks for the different TCGA tumor entities at the ccRCC-enhancer site with gained activity in ccRCC . KIRC specific ATAC-seq sites 10767-70 are indicated above the tracks. d Spearman’s rank correlation analysis of HIF-2α mRNA expression (TPM: transcripts per million) and accessibility at KIRC_10770 in the KIRC data set. Values are from samples for which both analyses were available ( n = 16 different tumors). Gray region depicts 95% confidence interval. Significance was assessed with a two-sided t-approximation test.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Sequencing, ChIP-sequencing, Activity Assay, Capture-C, Comparison, Expressing

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a RSEM-normalized RNA-seq values for HIF-2α in the TCGA KIRC cohort stratified for VHL copy number alterations (diploid n = 38, deletion n = 309). P -values were determined by an unpaired, two-tailed t-test. b RT-qPCR analyses for HIF-2α mRNA in RCC4 cells with or without functional pVHL. Graph shows mean + SD. P -values were determined by a two-tailed one sample t-test with a hypothetical value of 1. n = 4 independent experiments. c Representative immunoblot ( n = 2) for HIF-1α, HIF-2α, hemagglutinin (HA), and β-actin in lysates from RCC4/i.VHL-HA cells exposed to 0.5 µg/ml doxycycline to induce pVHL for the indicated time. d RT-qPCR analyses for HIF-2α mRNA in lysates from RCC4/i.VHL-HA cells treated with 0.5 µg/ml doxycycline for the indicated time. Expression values are mean + SD from three independent experiments. Significance was tested by a two-tailed one sample t-test with a hypothetical value of 1. e Expression qPCR analysis for HIF-2α mRNA in lysates from RCC4 cells transfected with sgRNA targeting HIF-1β or non-targeting (nt) control using CRISPR/Cas9. Mean + SD from 7 independent experiments. Differences were assessed by a two-tailed one sample t-test with a hypothetical value of 1. f RT-qPCR analysis for HIF-2α mRNA in lysates from 786-0 cells transfected with sgRNA targeting HIF-1β or non-targeting (nt) control using CRISPR/Cas9. Graph shows mean + SD from 5 independent experiments. P -values were determined by a two-tailed one sample t-test with a hypothetical value of 1. g Representative immunoblot ( n = 2) for HIF-2α, HIF-1α, HIF-1β, and β-actin from lysates of RCC4 cells transfected with sgRNA targeting HIF-1β or non-targeting (nt) control using CRISPR/Cas9. h Representative immunoblot ( n = 2) for HIF-2α, HIF-1β, and β-actin from lysates of 786-0 cells transfected with sgRNA targeting HIF-1β or non-targeting (nt) control using CRISPR/Cas9. i ATAC-seq tracks at the ccRCC-activated enhancer in RCC4/i.VHL-HA cells as well as single clones of HIF-1β knock-out or nt control RCC4 or 786-0 cells, respectively. RCC4/i.VHL-HA were exposed either to 0.5 µg/ml doxycycline (Dox +) or to DMSO control (Dox -) for 18 h. The ATAC element KIRC_10770 is highlighted in orange. j H3K27ac CUT&Tag tracks from the same cells at the same genomic region as in i ).

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: RNA Sequencing, Two Tailed Test, Quantitative RT-PCR, Functional Assay, Western Blot, Expressing, Transfection, Control, CRISPR, Clone Assay, Knock-Out

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Expression qPCR analyses for HIF-2α mRNA in lysates from 105 isolates of primary tubule cells (PTC) exposed to 1 mM DMOG or control conditions for 16 h. Values are mean +/− SD from n = 105 independent experiments. Unpaired, two-tailed t-test. b Expression qPCR for HIF-2α mRNA in lysates from PTC depleted for HIF-1α or HIF-1β using siRNA and stimulated with or without 1 mM DMOG for 16 h. Non-targeting (nt) control siRNA was used as control. Values are mean + SD. Significance was tested by two-way ANOVA followed by Bonferroni’s post hoc test. Data is from nine independent experiments using PTC from nine different individuals. c HIF ChIP-seq tracks from lysates of primary tubule cells (PTC, green) treated with 1 mM DMOG for 4 h and ccRCC cells (blue) at the PRKCE-EPAS1 locus. ChIP-seq track for the activity marker H3K27ac in isolated ccRCC cells from patient #1 and hypomethylated CpGs in TCGA KIRC are also shown. KIRC-specific polymorphisms identified by the Cis-eQTL PancanQTL analysis as an eQTL for EPAS1 expression are indicated . Germline variants associated with renal cancer development within the EPAS1 -coding region are shown (RCC risk SNP) , . d Correlation of rs12617313 genotype and HIF-2α mRNA expression in the KIRC TCGA normal tissue and tumor data. Number of samples for normal tissue: Genotype TT n = 19, AT n = 25, AA n = 16. Number of samples for tumor tissue (KIRC): Genotype TT n = 94, AT n = 188, AA = 121. Data shown are median and interquartile range. χ 2 -test; p -value as indicated for higher expression in AA individuals compared with TT individuals. e Genotype-expression correlation for rs12617313 and HIF-2α mRNA in PTC exposed to 1 mM DMOG for 16 h or left untreated. Number of samples for each genotype: TT n = 26, AT n = 58, AA n = 21. Data shown are median and interquartile range. Significance was tested by two-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Expressing, Control, Two Tailed Test, ChIP-sequencing, Activity Assay, Marker, Isolation

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a ChIP-seq tracks for HIF-1β, PAX8 , H3K27ac and HNF-1β CUT&Tag-seq track from 786-0 cells at EPAS1 -enhancer E2. Binding sites for HNF-1β (E2_HNF-1β) and PAX8 (E2_PAX8) within KIRC_10767 are highlighted in blue. HIF-binding site is marked in orange. b Expression qPCR analyses for HIF-2α mRNA in lysates from 786-0 cells depleted for the indicated transcription factor (HNF-1β or PAX8). Values are mean + SD from 5 (HNF-1β) or 4 (PAX8) independent experiments. Statistical significance was assessed by a two-tailed one sample t-test with a hypothetical value of 1. c Immunoblot analyses for HIF-2α, HNF-1β, and β-actin in lysates from 786-0 cells depleted for HNF-1β using CRISPR/Cas9. Representative blot from two independent experiments with similar results. d Immunoblot analyses for HIF-2α, PAX8, and β-actin in lysates from 786-0 cells depleted for PAX8 using CRISPR/Cas9. Representative blot from two independent experiments with similar results. e ATAC-seq and CUT&Tag-seq tracks of the chromatin activity marker H3K27ac from single clones of HNF-1β knock-out, PAX8 knock-out or control (nt) clones of 786-0 cells at EPAS1 -enhancer E2. The ATAC KIRC element 10767 harboring binding sites for HNF-1β and PAX8 is highlighted in blue, the HIF-1β binding site at KIRC_10770 is marked in orange. f Schematic depiction of transcription factor binding sites for HNF-1β, PAX8 and HIF-1β at EPAS1- enhancer E2. Positions of sgRNAs used for knock-out of the respective binding sites are indicated in orange. Created in BioRender. Naas, S. (2025) https://BioRender.com/w24j560 . g Expression qPCR analysis for HIF-2α mRNA in pools of 786-0 cells transfected with sgRNA targeting the HNF-1β (E2_HNF-1β) or PAX8 (E2_PAX8) binding site at the EPAS1- enhancer E2. Cells treated with non-targeting sgRNA served as controls. Values are mean + SD. p -values were determined by a two tailed one sample t-test with a hypothetical value of 1. n = 5 (E2_HNF-1β) or n = 6 (E2_PAX8) independent experiments. h Immunoblot analyses for HIF-2α and β-actin in 786-0 cells with intact (nt) or mutated binding sites for HNF-1β (E2_HNF-1β) or PAX8 (E2_PAX8) at the EPAS1 -enhancer E2. Representative blot from two independent experiments with similar results.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: ChIP-sequencing, Binding Assay, Expressing, Two Tailed Test, Western Blot, CRISPR, Activity Assay, Marker, Clone Assay, Knock-Out, Control, Transfection

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Schematic of the two HIF-binding EPAS1 -enhancers E1 and E2 at the PRKCE-EPAS1 locus. The location of HIF-binding motifs and positions of sgRNAs within the KIRC elements are indicated. Created in BioRender. Naas, S. (2025) https://BioRender.com/w24j560 . b Expression qPCR for HIF-2α mRNA in lysates from primary ccRCC cells isolated from 4 different tumor nephrectomies transfected with a pool of sgRNAs directed against the HIF-binding-sites at EPAS1 -enhancer 1 (E1) and EPAS1 -enhancer 2 (E2_HIF) or a control sgRNA (nt). Values are mean + SD from 4 independent experiments. Significance was determined by a two tailed one sample t-test with a hypothetical value of 1. Expression qPCR for HIF-2α mRNA in lysates from 786-0 ( c ) or RCC4 ( d ) cells transfected with sgRNAs targeting EPAS1 -enhancer 1 (E1), the HIF-binding site of EPAS1 -enhancer 2 (E2_HIF) or both enhancers (E1 + E2_HIF). nt = non-targeting control sgRNA. Values are mean + SD from independent experiments (786-0: n = 7 for nt; n = 6 for E1 and E2_HIF; n = 5 for E1 + E2_HIF; RCC4: n = 4 for nt and E1 + E2 HIF; n = 3 for E1 and E2_HIF). Significance was tested by a two tailed one sample t-test with a hypothetical value of 1. Immunoblot analyses for HIF-2α and β-actin protein in lysates from 786-0 ( e ) or RCC4 ( f ) cells transfected with sgRNAs targeting the enhancers as indicated. Representative blot from three (786-0) or two (RCC4) independent experiments with similar results. Gene set enrichment analysis for differentially expressed genes comparing RNA-seq data generated in 786-0 ( g ) or RCC4 ( h ) cells transfected with sgRNA targeting E2_HIF or non-targeting control guides (nt). One RNA sample of each condition was sequenced in technical duplicates. i Comparison of log 2 (fold change) expression values of genes significantly regulated upon E2_HIF knock-out in 786-0 or RCC4 cells. HIF-target transcripts NDRG1, VEGFA, and CCND1 are indicated. One RNA sample of each condition was sequenced in technical duplicates.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Binding Assay, Expressing, Isolation, Transfection, Control, Two Tailed Test, Western Blot, RNA Sequencing, Generated, Comparison, Knock-Out

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Representative immunoblot ( n = 3) for HIF-2α and β-actin protein in lysates from single clones of EPAS1 -enhancer 2 defective (E2_HIF) or control (non-targeting, nt) 786-0 cells. b Xenograft tumor assay in NOD/SCID-gamma mice using EPAS1 -enhancer 2 defective (E2_HIF ko) or intact (nt) clones of 786-0 cells. Volume values are mean ± SD from 6 tumors per cell clone. Two-way ANOVA followed by Bonferroni’s post hoc test comparing tumor volume between mean nt and E2_HIF ko at the indicated time points. Significant differences (** p < 0.01) were detected for comparisons between mean values of E2_HIF ko (2.1 or 2.2) with nt (0.1 or 0.2) xenografts for weeks 8, 9 and 10. c Weight of the explanted xenograft tumors. Data shown are median and interquartile range from 5 (E2_HIF 2.1) or 6 (nt 0.1, nt 0.2, E2_HIF2.2) tumors. One-way ANOVA followed by Bonferroni’s post hoc test. d Volcano plot from RNA-seq experiments in E2_HIF ko versus nt clones of 786-0 cells. HIF-2α mRNA as well as HIF-target transcripts NDRG1, VEGFA, and CCND1 are indicated. Data is from the two clones of cells per condition from a) and b). e GSEA for HIF-binding sites in 786-0 cells and RNA-seq data ( n = 2 clones) from d). DEG: differentially expressed genes, ES: enrichment score. p -value was estimated using adaptive multilevel Monte Carlo scheme and adjusted for multiple testing with the Benjamini–Hochberg method. f CUT&Tag-seq (C&T) for HIF-1β and H3K27ac as well as ATAC-seq at EPAS1 -enhancer 2 and the control locus EGLN3 from E2_HIF ko versus nt clones of 786-0 cells. g CUT&Tag-seq signals for HIF-1β or H3K27ac integrated over 543 ChIP-seq pre-defined HIF1-β binding sites in E2_HIF ko versus nt clones of 786-0 cells. Non-HIF binding enhancers served as control. Signals were centered on the H3K27ac peak within the enhancers. h Comparison of log 2 (fold change) expression values of genes significantly regulated in E2_HIF ko 786-0 cells ( n = 2 clones) or through HIF-2 inhibition by PT2385 in 786-0 cells . HIF-target transcripts NDRG1, VEGFA, and CCND1 are indicated. i The transcriptional circuitry of HIF-2α expression at the EPAS1 gene locus. Created in BioRender. Naas, S. (2025) https://BioRender.com/w24j560 .

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Western Blot, Clone Assay, Control, RNA Sequencing, Binding Assay, ChIP-sequencing, Comparison, Expressing, Inhibition

Journal: Nature Communications

Article Title: Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility

doi: 10.1038/s41467-019-12097-6

Figure Lengend Snippet: Divergent hIgG agonism and specific FcγR-binding requirement. a Diagram showing the OVA-specific CD8 + T-cell response model. In brief, FcγR-humanized (hFCGR Tg ) or -deficient (FcγRα −/− ) mice were adoptively transferred with OT-I cells on day-1, immunized intraperitoneally (i.p.) with 2 μg of DEC-OVA in the presence of control or anti-CD40 antibodies on day 0. Splenocytes were harvested to quantify OVA-specific CD8 + T cells on day 6. b , c Representative FACS profile ( b ) and quantification ( c ) showing the percentage of OT-I cells (CD45.1 + TCRVα2 + ) among CD8 + T cells in mice treated and analyzed as in ( a ) together with 30 μg of indicated control or anti-mCD40 antibodies. Numbers of mice: b , c three hFCGR Tg mice for Ctrl IgG, five hFCGR Tg , and three FcγRα −/− mice for other groups. d – f Quantification of OT-I cells as the percentage of OT-I cells among CD8 + T cells ( d , e ) or cell count ( f ) in FcγR-humanized mice treated and analyzed as in ( a ) together with 10 μg ( d , e ) or 30 μg ( f ) of indicated control or anti-mCD40 antibodies (the N297A mutation abrogates Fc–FcγR binding) and with/without FcγRIIB-blocking antibody 2B6 (150 μg per mouse) ( f ). Numbers of mice: d four mice per group; e five mice per group; f two mice for Ctrl IgG, five mice for αmCD40:G2, six mice for αmCD40:G2 + 2B6. g Quantification of OT-I cells as the percentage of OT-I cells among CD8 + T cells in mice of indicated genotypes (FcγR-deficient (FcgRα −/− , five mice per group), FcγRIIB-deficient (Fcgr2b −/− , five mice per group) or humanized (Fcgr2b −/− hFCGR2B Tg , four mice per group)) treated and analyzed as in ( a ) together with 10 μg of indicated control or IgG2 anti-mCD40 antibodies. Each symbol represents an individual mouse. Bars represent the mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; unpaired two-tailed t test ( c , g ), one-way ANOVA with Holm–Sidak’s post hoc ( d – f ). Source data ( c – g ) are provided as a Source Data file. A representative of two independent experiments is shown

Article Snippet: Serially diluted control IgG or anti-CD40 antibodies (3.16–0.00316 μg ml −1 ) were then added and incubated for 1 h. After washing for three times, horseradish peroxidase-conjugated detection antibody (anti-human IgG Fc-HRP, Bethyl Laboratories, 100 ng ml −1 ) was added and incubated for 1 h. The plate was then washed and developed with TMB peroxidase substrate (KPL) in the dark for 20–40 min.

Techniques: Binding Assay, Control, Cell Counting, Mutagenesis, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility

doi: 10.1038/s41467-019-12097-6

Figure Lengend Snippet: CH1-hinge basis of agonistically inactive IgG3 and superior IgG2. a – c Quantification of OT-I cells as the percentage of OT-I cells among CD8 + T cells ( a ) or cell count ( b , c ) in FcγR-humanized mice treated and analyzed as in Fig. together with indicated control or anti-mCD40 antibodies ( a 3.16 μg per mouse; b , c 10 μg per mouse). Numbers of mice: a six mice for Ctrl IgG, six to seven mice per group for others; b , c three mice for Ctrl IgG, four to five mice per group for others. d – f Representative FACS profile ( d ) and quantification ( e , f ) showing the percentage of OT-I cells (CD45.1 + TCRVα2 + ) among CD8 + T cells in human CD40/FcγR-transgenic mice treated and analyzed as in Fig. together with control or anti-human CD40 antibodies of indicated clones (Clones 21.4.1 and 3.1.1 have been described in Patent No.:US 7,338,660; Clone 21.4.1 in the IgG2 form is also known as CP-870,893) and constant domains (30 μg per mouse). Numbers of mice: d – f five to six mice per group. Each symbol represents an individual mouse. Bars represent the mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; one-way ANOVA with Holm–Sidak’s post hoc. Source data ( a – c , e , f ) are provided as a Source Data file. A representative of two independent experiments is shown

Article Snippet: Serially diluted control IgG or anti-CD40 antibodies (3.16–0.00316 μg ml −1 ) were then added and incubated for 1 h. After washing for three times, horseradish peroxidase-conjugated detection antibody (anti-human IgG Fc-HRP, Bethyl Laboratories, 100 ng ml −1 ) was added and incubated for 1 h. The plate was then washed and developed with TMB peroxidase substrate (KPL) in the dark for 20–40 min.

Techniques: Cell Counting, Control, Transgenic Assay, Clone Assay

Journal: Nature Communications

Article Title: Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility

doi: 10.1038/s41467-019-12097-6

Figure Lengend Snippet: Impact of both IgG CH1-hinge and Fc on anti-CD40 antibody antitumour activities. MC38 ( a , b ) and MO4 ( c ) tumour volumes in FcγR-humanized mice following treatment with control or anti-mCD40 antibodies of indicated constant domains. After tumour cells were subcutaneously inoculated and established in FcγR-humanized mice, mice were treated i.p. twice on day 0 (the day when mice with palpable tumours receive their first treatment) and day 3 with 31.6 μg/mouse of control or anti-CD40 antibodies of indicated constant domains (the N297A mutation abrogates Fc–FcγR binding), and monitored for tumour growth. For mice inoculated with MO4 tumour cells in ( c ), each treatment also included 2 μg/mouse of DEC-OVA. Shown are tumour growth curves of individual mice ( a ) or mouse groups ( b , c ). Numbers of mice: a , b seven to eight mice per group; c seven mice per group except six mice for αmCD40:G2(N297A). Bars represent the mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, chi-square test ( a ), and two-way ANOVA with Holm–Sidak’s post hoc ( b , c ) were used for group comparison. Source data ( a – c ) are provided as a Source Data file. A representative of two independent experiments is shown ( a , b )

Article Snippet: Serially diluted control IgG or anti-CD40 antibodies (3.16–0.00316 μg ml −1 ) were then added and incubated for 1 h. After washing for three times, horseradish peroxidase-conjugated detection antibody (anti-human IgG Fc-HRP, Bethyl Laboratories, 100 ng ml −1 ) was added and incubated for 1 h. The plate was then washed and developed with TMB peroxidase substrate (KPL) in the dark for 20–40 min.

Techniques: Control, Mutagenesis, Binding Assay, Comparison

Journal: Nature Communications

Article Title: Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility

doi: 10.1038/s41467-019-12097-6

Figure Lengend Snippet: The superior rigidity of IgG2 CH1-hinge. a A diagram showing anti-mCD40 antibody TR-FRET. Anti-mCD40 antibody molecules mixed with CD40-Tb and CD40-D2 (mCD40 labeled with Tb donor and D2 acceptor fluorochromes, respectively) can simultaneously bind CD40-Tb and CD40-D2, and emit TR-FRET signal quantified as the relative ratio of detected 665 nm fluorescence to 620 nm fluorescence (Em665/Em620) upon stimulation. b A diagram of the model showing that hinge flexibility of hIgG anti-mCD40 antibodies correlates with TR-FRET signal levels. Left, anti-mCD40 antibodies with little hinge flexibility do not trigger TR-FRET signal due to the large distance between CD40-Tb and CD40-D2; middle, hinge flexibility can bring CD40-Tb and CD40-D2 close enough to trigger TR-FRET signal; right, anti-mCD40 antibodies with large hinge flexibility give rise to stronger TR-FRET signal due to more molecules with closer CD40 binding sites. c – e TR-FRET signal levels of anti-mCD40 antibodies of indicated constant domains. Shown are relative TR-FRET signal levels (Em665/Em620) plotted against the concentration of control IgG or the anti-mCD40 antibodies of indicated constant domains. Bars represent the mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, two-way ANOVA with Holm–Sidak’s post hoc. Source data ( c – e ) are provided as a Source Data file. A representative of two independent experiments is shown

Article Snippet: Serially diluted control IgG or anti-CD40 antibodies (3.16–0.00316 μg ml −1 ) were then added and incubated for 1 h. After washing for three times, horseradish peroxidase-conjugated detection antibody (anti-human IgG Fc-HRP, Bethyl Laboratories, 100 ng ml −1 ) was added and incubated for 1 h. The plate was then washed and developed with TMB peroxidase substrate (KPL) in the dark for 20–40 min.

Techniques: Labeling, Fluorescence, Binding Assay, Concentration Assay, Control

Journal: Frontiers in Immunology

Article Title: Fc-Galactosylation of Human Immunoglobulin Gamma Isotypes Improves C1q Binding and Enhances Complement-Dependent Cytotoxicity

doi: 10.3389/fimmu.2017.00646

Figure Lengend Snippet: Increased C1q binding of Fc-galactosylated rituximab-derived immunoglobulin gamma 1 (IgG1) and IgG3. Kinetic of C1q binding to CD20 + Raji cells in the presence of galactosylated or degalactosylated glycovariants of human IgG1–4. Exemplary C1q-binding curves (left), flow cytometry histograms of C1q binding after 60 min (center) and statistics at time point 60 min of at least three independent experiments. Statistical analysis: unpaired two-tailed Student’s t -test, mean ± SEM, ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001. MFI, median fluorescence intensity.

Article Snippet: Monoclonal mouse anti-human C1q (Cat. No.: A201) and biotinylated anti-human C1q (Cat. No.: A700) were purchased from Quidel ® Corporation.

Techniques: Binding Assay, Derivative Assay, Flow Cytometry, Two Tailed Test, Fluorescence

Journal: bioRxiv

Article Title: Post-Transplant Administration of G-CSF Impedes Engraftment of Gene Edited Human Hematopoietic Stem Cells by Exacerbating the p53-Mediated DNA Damage Response

doi: 10.1101/2023.06.29.547089

Figure Lengend Snippet: a, Experimental scheme. Pre-cultured human MPB CD34+ cells were electroporated with Cas9 alone (Cas9 group), AAVS1-specific sgRNA/Cas9 RNP (RNP group), or RNP in the presence of GSE56 mRNA (RNP/GSE56 group). Edited CD34+ cells were further cultured for 4 hours in the presence or absence of G-CSF at a dose of 100 ng/mL. Expression of p21 and DNA damage response (DDR) sensors were measured by RT-qPCR and confocal microscopy, respectively, at 28 hours post-electroporation. b , Relative p21 gene expression, as measured by RT-qPCR (n = 15 technical replicates/group, from 5 independent donors). c , Representative confocal images of DAPI (blue) and DNA damage response sensor 53BP1 (green) within each group of human CD34+ HSPCs. Asterisks indicate 53BP1 foci. Scale bar represents 10 µm. d , Average numbers of 53BP1 foci per cell (n = 844-1248 cells/group, from 3 independent donors). e , Representative confocal images of DAPI (blue) and DNA damage response sensor γH2AX (red) within each group of human CD34+ HSPCs. Asterisks indicate γH2AX foci. Scale bar represents 10 µm. f , Average numbers of γH2AX foci per cell (n = 844-1248 cells/group, from 3 independent donors). In panel b , data are displayed as mean ± standard error of the mean (SEM) and one way ANOVA with Tukey’s multiple comparison test was used. In panels d and f , data are displayed as mean ± SEM and Kruskal-Wallis test with Dunn’s multiple comparison test was used. ns, not significant, * p ≤ 0.05, **** p ≤ 0.0001.

Article Snippet: Cells were then stained with rabbit anti-53BP1 antibody (Bethyl Laboratories, Montgomery, TX, USA, A700-011) and mouse anti-phospho- histone H2A.X (Ser139) antibody (MilliporeSigma, Burlington, MA, USA, 05-636) in 1:200 dilution at 4°C overnight.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Confocal Microscopy, Electroporation, Gene Expression, Comparison

Journal: eLife

Article Title: The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells

doi: 10.7554/eLife.70555

Figure Lengend Snippet: ( a ) Body weight of naive mice was determined weekly. ( b ) At 12 weeks of age, male and female mice were examined for rectal prolapse. ( c ) Splenomegaly and colon length summarized in ( d ). ( e ) Representative gross autopsy of a hemorrhagic abdomen, ( f–i ) Lymphoid cells from spleen, mesenteric (mLN), peripheral (pLN) lymph nodes, or the small intestine lamina propria were collected from Foxp3 cre and PHD2 ΔTreg mice. ( f ) Frequency of conventional, Foxp3- CD4 and CD8-expressing cells among TCRβ-expressing T lymphocytes. ( g ) Representative merged (n = 15) t-distributed stochastic neighbor embedding (t-SNE) plot after dimensionality reduction and unsupervised clustering of flow cytometry data from CD4-expressing spleen cells. Relative distributions of CD4 + lymphocyte subsets are shown as doughnut charts. ( h ) Frequency of effector-like (CD44 hi CD62L lo ) conventional T lymphocytes in the indicated lymphoid organs. ( i ) Frequency of IFN-γ (top panel) and IL-17A (bottom panel) producing CD4 + T cells after in vitro stimulation. ( j ) Expression of inflammatory cytokines determined by qPCR on extracts from unfractionated mLNs. Data are representative of at least three independent experiments with n = 9 ( a, j ), n = 25 ( d ), and n = 15 ( f–i ) per group. Values are presented as the mean ± SD and were compared by two-tailed unpaired Student’s t- test. Only significant differences are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Naive Tconv: Foxp3 - CD44 - CD62L + ; Memory Tconv: Foxp3 - CD44 + CD62L + ; Effector Tconv: Foxp3 - CD44 + CD62L - ; Memory Treg: Foxp3 + CD44 + CD62L + ; Effector Treg: Foxp3 + CD44 + CD62L - . Figure 1—source data 1. PHD2 ΔTreg mice display a spontaneous Th1-like inflammatory syndrome.

Article Snippet: Antibody , Anti-mouse CD62L-A700 (MEL-14, mouse monoclonal) , BD Biosciences , 560517;RRID: AB_1645210 , (1:100).

Techniques: Expressing, Flow Cytometry, In Vitro, Two Tailed Test

Journal: eLife

Article Title: The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells

doi: 10.7554/eLife.70555

Figure Lengend Snippet: Foxp3 cre and PHD2 ΔTreg male mice were provided with 2% DSS in tap water for 5 days. On day 5, the 2% DSS water was replaced with normal drinking water and mice were followed during 14 days for ( a ) body weight, ( b ) survival, ( c ) colitis severity, and ( d ) colon length. ( e ) Colons were isolated from untreated mice or 6 days after colitis induction and were fixed and stained with hematoxylin and eosin (H&E); arrows indicate inflammatory cell infiltrates. ( f ) Foxp3 cre and PHD2 ΔTreg male mice were infected by intragastric gavage with 10 cysts of ME-49 type II Toxoplasma gondii (control group are Foxp3 cre mice without treatment) and subsequently followed for ( g ) body weight. ( h ) Mice were sacrificed 8 days after infection to assess colon length. ( i ) Frequency of effector-like (CD44 hi CD62L lo ) conventional T lymphocytes in the indicated lymphoid organs. ( j ) Frequency of IFN-γ-producing CD4 + T cells after in vitro stimulation. ( k ) Frequency of T-bet + among Foxp3 + (Treg) cells. Data are representative of three independent experiments with n = 20 ( a, b ), n = 10–14 ( c, d ), n = 5 ( e ), or n = 10 ( g–k ) per group. Values are presented as the mean ± SD and were compared by two-tailed unpaired Student’s t -test ( a, c, g ), by Mantel–Cox test ( b ), one-way ANOVA with Tukey’s multiple comparisons test ( d, h ) or two-way ANOVA with Tukey’s multiple comparisons test ( i–k ). Only significant differences are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 5—source data 1. Increased sensitivity of PHD2 ΔTreg mice to dextran sodium sulfate (DSS)-induced colitis and toxoplasmosis.

Article Snippet: Antibody , Anti-mouse CD62L-A700 (MEL-14, mouse monoclonal) , BD Biosciences , 560517;RRID: AB_1645210 , (1:100).

Techniques: Isolation, Staining, Infection, In Vitro, Two Tailed Test

Journal: eLife

Article Title: The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells

doi: 10.7554/eLife.70555

Figure Lengend Snippet: ( a ) Representative gross autopsy of spleens and colon length summarized in ( b ) of Foxp3 cre , PHD2 ΔTreg , PHD2-HIF1α ΔTreg , PHD2-HIF2α ΔTreg , and PHD2-HIF1α-HIF2α ΔTreg (TKO) mice. ( c ) Representative merged (n = 15) t-distributed stochastic neighbor embedding (t-SNE) plot after dimensionality reduction and unsupervised clustering of flow cytometry data from CD4-expressing spleen cells. Relative distributions of CD4 + lymphocyte subsets are shown as doughnut charts. ( d–g ) Lymphoid cells from spleen, mesenteric (mLN), peripheral (pLN) lymph nodes, or the small intestine lamina propria were collected from Foxp3 cre , PHD2 ΔTreg , PHD2-HIF1α ΔTreg mice, PHD2-HIF2α ΔTreg and PHD2-HIF1α-HIF2α ΔTreg (TKO) male and female mice and the relative frequency and phenotype of Foxp3-positive and Foxp3-negative, conventional T lymphocytes determined by flow cytometry. ( d ) Frequency of effector-like (CD44 hi CD62L lo ) conventional T lymphocytes in the indicated lymphoid organs. ( e ) Frequency of IFN-γ-producing CD4 + T cells after in vitro stimulation. ( f ) Frequency of Foxp3-expressing cells among CD4-positive T lymphocytes. ( g ) Ratio of the Foxp3 MFI of PHD2-KO, PHD2-HIF1αKO, PHD2-HIF2αKO, or TKO splenic Tregs to Foxp3 cre splenic Tregs. Data are representative of at least three independent experiments with n = 15 per groups. Values are expressed as the mean ± SD and were compared by one-way ANOVA with Tukey’s multiple comparisons test ( b, g ) or two-way ANOVA with Tukey’s multiple comparisons test ( d–f ). Only significant differences are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 6—source data 1. Concomitant loss of HIF2α but not HIF1α expression attenuates the proinflammatory phenotype of PHD2 ΔTreg mice.

Article Snippet: Antibody , Anti-mouse CD62L-A700 (MEL-14, mouse monoclonal) , BD Biosciences , 560517;RRID: AB_1645210 , (1:100).

Techniques: Flow Cytometry, Expressing, In Vitro

Journal: eLife

Article Title: The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells

doi: 10.7554/eLife.70555

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-mouse CD62L-A700 (MEL-14, mouse monoclonal) , BD Biosciences , 560517;RRID: AB_1645210 , (1:100).

Techniques: Recombinant, Sequencing, Software