Journal: bioRxiv

Article Title: Aging restricts maturation of CXCL13 + T follicular helper cells in human immunity

doi: 10.64898/2026.04.03.715992

Figure Lengend Snippet: a, Schematics of key differences in Tfh cell maturation between humans (left) and mice (right). b, Gene expression signatures of all CD4 + T cell clusters from tonsillar T cell scRNA-seq. c, Volcano plot showing differential gene expression of GC-Tfh cells (left) vs. CXCL13 + GC-Tfh cells (right). d, Select subset-specific marker gene expression in Tfh, GC-Tfh, and CXCL13 + GC-Tfh cells. e, Schematic overview for assessment of CXCL13 production by non-, pre-, and GC-Tfh cells. f, Exemplary gating for non-Tfh (PD-1 - CXCR5 - ), pre-Tfh (PD-1 lo CXCR5 lo ), and GC-Tfh cells (PD-1 hi CXCR5 hi ). g, CXCL13 secretion by different sorted T cell subtypes cultured with or without B cells (n = 4). h, Schematic representation for CRISPR/Cas9-based editing of tonsil samples. i, CXCL13 ELISA of tonsillar cell culture supernatants for indicated transcription factor knockouts vs. scramble control (n = 14; ≤ 30 yo). Data were analyzed using two-way ANOVA with Šídák’s multiple comparison test ( g ) and one-way ANOVA with Dunnett’s multiple comparisons test ( i ). * P < 0.05, ** P < 0.01.

Article Snippet: Editing efficiencies ( Table S3 ) were calculated using the Inference of CRISPR Edits (ICE) analysis tool (Synthego Performance Analysis 2019, v3.0) to ensure target gene knockout.

Techniques: Gene Expression, Marker, Cell Culture, CRISPR, Enzyme-linked Immunosorbent Assay, Control, Comparison

Journal: bioRxiv

Article Title: Aging restricts maturation of CXCL13 + T follicular helper cells in human immunity

doi: 10.64898/2026.04.03.715992

Figure Lengend Snippet: a, Number of differentially expressed genes (DEGs, >50yo vs. <30yo) over pseudotime during Tfh cell development from naïve CD4 + T cells (left) to CXCL13 + GC-Tfh cells (right). False discovery rate (FDR) is set to < 0.05. b, Volcano plot showing differential gene expression of Tfh cells from older (left) vs. younger donors (right). c, Schematic representation for CRISPR/Cas9-based editing of naïve CD4 + T cells followed by T cell polarization and flow cytometric analysis. d, Expression of CXCR5, FOXP3, and PD-1 in naïve CD4 + T cells cultured under Tfh-polarizing conditions (dark grey) or non-polarizing conditions (light grey). e, Log2 fold change relative to matched scramble controls for Th1 (left) and Th17 (right) cell frequency in TF-knockouts under Th1- or Th17-polarizing conditions. f, Log2 fold change relative to matched scramble controls for Tfh cell frequency in TF-knockouts under Tfh-polarizing conditions (gated on CD4 + CD19 - FOXP3 - CXCR5 + PD-1 + cells). Data were analyzed using a mixed effects model and Dunnett’s multiple comparisons test ( f ), one sample t test ( e ). Error bars SEM ( e-f ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Editing efficiencies ( Table S3 ) were calculated using the Inference of CRISPR Edits (ICE) analysis tool (Synthego Performance Analysis 2019, v3.0) to ensure target gene knockout.

Techniques: Gene Expression, CRISPR, Expressing, Cell Culture

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: Knock-out of enhancer regions in cell pools was evaluated using the “Inference of Crispr Editing” method (ICE v2 CRISPR Analysis Tool, Synthego, Menlo Park, CA, USA).

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 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: Knock-out of enhancer regions in cell pools was evaluated using the “Inference of Crispr Editing” method (ICE v2 CRISPR Analysis Tool, Synthego, Menlo Park, CA, USA).

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