Journal: bioRxiv

Article Title: TGFβ signaling systems are prone to inhibition and ligand competition by coreceptor

doi: 10.64898/2026.03.01.708909

Figure Lengend Snippet: Dependence of prevalent coreceptor trends on additional ligand characteristics and biochemical validation of engineered ENG -/- Tgfbr3-HA MCF10A cells. Related to . (A) Low-affinity ligands with high k rD for A (≡ R II ) favor the +,– trend and disfavor the – trend. (B) Ligands with higher affinity for B (≡ R I ) than for C (≡ coreceptor) favor the +,– trend. (C) Immunoblot of parental MCF10A-5E cells (+/+) and FACS sorted MCF10A-5E ENG -/- cells (–/–) for ENG with tubulin and p38 used as loading controls. (D) Immunoblot of MCF10A-5E ENG -/- stably transduced with pSLIK-Tgfbr3-HA, treated with or without 1 µg/ml doxycycline (DOX) for 24 hours, and probed for Tgfbr3 and HA tag with tubulin and ERK1/2 used as loading controls. (E) Representative biplots of Tgfbr3-HA and phospho (P-)/total SMAD2 for ENG -/- Tgfbr3-HA MCF10A cells treated with or without 1 µg/ml DOX for 24 hours and 3 ng/ml TGFβ1 for 30 minutes. The bootstrapped means for each decile are overlaid. For (A) and (B), differences between the observed (bars) and expected (gray) distributions were assessed by K-S test. ∼ 0 is <10 -300 .

Article Snippet: Lentivirus was prepared in HEK293T/17 cells (ATCC) by triple transfection of pSLIK-Tgfbr3-HA neo with psPAX2 + pMD.2G (Addgene) and transduced into ENG -/- MCF10A-5E cells as described previously.

Techniques: Biomarker Discovery, Western Blot, Stable Transfection, Transduction

Journal: bioRxiv

Article Title: Near completely reversing the γ- to β-globin switch by enhancer release, retargeting and reinforcing

doi: 10.64898/2026.01.30.702713

Figure Lengend Snippet: a The results of the CRISPR–Cas9 screen showing enriched sgRNAs in HUDEP-2 cells expressing high levels of HbF. The y-axis shows the log 2 (fold-change (FC)) in sgRNAs. The sgRNAs targeting the homologous sequences of HBB and HBD (blue dots), the proximal CACCC motif (red dot) and the distal CACCC motif (orange dot) in HBB are highlighted. b HbF levels of individuals with mutations in the CACCC motif in the HBB promoter in the Globin Gene Server. c Cas9-expressing HUDEP-2 cells were transduced with sgRNAs targeting the CACCC motif in HBB . The average editing efficiencies of PM-1, PM-2 and DM were 79.1%, 67.4% and 45.4%, respectively. The charts show β-like globin gene expression relative to β-actin mRNA expression as measured by RT–qPCR (mean ± s.d., n = 3). Multiple comparisons were assessed with one-way ANOVA with Tukey’s MCT. * P < 0.05, ** P < 0.01 and *** P < 0.001. d Chromosome conformation capture analysis of control and CACCC motif edited HUDEP-2 cells (mean ± s.d., n = 3). e The relative frequencies of the transcriptional bursts of HBB and HBG in HUDEP-2 clones were tested by Chromium single cell sequencing. f ATAC-seq signals at the β-globin cluster were analyzed in control and CACCC motif edited HUDEP-2 cells, along with CUT&Tag enrichment for CTD, H3K4me3, H3K9Ac, and H3K27Ac.

Article Snippet: A total of 3,784 sgRNAs were designed targeting the downstream region of HBG (hg38/GRCh38, chr11: 5204054-5248601) , followed by the generation of a pooled lentiviral vector library using the lentiGuide-Puro plasmid (Addgene, plasmid 52963) that contains a puromycin-resistance cassette for selection.

Techniques: CRISPR, Expressing, Transduction, Gene Expression, Quantitative RT-PCR, Control, Clone Assay, Single Cell, Sequencing

Journal: bioRxiv

Article Title: Near completely reversing the γ- to β-globin switch by enhancer release, retargeting and reinforcing

doi: 10.64898/2026.01.30.702713

Figure Lengend Snippet: a Cas9-expressing HUDEP-2 cells were transfected with sgRNAs targeting the TGACCA motif in HBG , followed by subsequent transfection with sgRNAs targeting the CACCC motif in HBB . b β-like globin gene expression relative to β-actin mRNA expression in HUDEP-2 cells from ( a ) as measured by RT–qPCR (mean ± s.d., n = 3). c β-like globin mRNA levels relative to β-actin mRNA levels in HUDEP-2 wild type (WT) clones (n = 7), GM clones (n = 14), PG clones (n = 6) and DG clones (n = 4) on day 5 of erythroid differentiation (mean ± s.d.). All GM, PG and DG clones carried four copies of HBG . d Fetal hemoglobin protein levels (normalized to total protein at 280 nm per 100 mAU*min) in four clonal populations from ( c ) as determined by HPLC. e Fetal Chromosome conformation capture analysis of HUDEP-2 cells from ( a ) (mean ± s.d., n = 3). f The relative frequency of the transcriptional bursts of HBB and HBG in HUDEP-2 clones from ( c ) were assessed by Chromium single cell sequencing. g ATAC-seq signals at the β-globin cluster were analyzed in HUDEP-2 cells from ( a ), along with CUT&Tag enrichment for CTD, H3K4me3, H3K9Ac, and H3K27Ac.

Article Snippet: A total of 3,784 sgRNAs were designed targeting the downstream region of HBG (hg38/GRCh38, chr11: 5204054-5248601) , followed by the generation of a pooled lentiviral vector library using the lentiGuide-Puro plasmid (Addgene, plasmid 52963) that contains a puromycin-resistance cassette for selection.

Techniques: Expressing, Transfection, Gene Expression, Quantitative RT-PCR, Clone Assay, Single Cell, Sequencing

Journal: Nature Immunology

Article Title: Spatial patterning of fibroblast TGFβ signaling underlies treatment resistance in rheumatoid arthritis

doi: 10.1038/s41590-025-02386-2

Figure Lengend Snippet: a – d , A fixed number of fibroblasts were seeded with varying numbers of HUVECs. Monoculture: 5,000 fibroblasts, 5:1 ratio: 5,000 fibroblasts and 1,000 HUVECs, 2:1 ratio: 5,000 fibroblasts and 2,500 HUVECs, 1:1 ratio: 5,000 fibroblasts and 5,000 HUVECs. a , b , ELISA quantification of POSTN ( a ) and COMP ( b ) production during co-culture. P values in the line graphs are displayed for the comparisons between monoculture and 1:1 ratio. The respective bar charts to the right represent the area under the curve. c , d , Flow cytometric quantification of fibroblast TGFβRII ( c ) and TGFβRIII ( d ) at the indicated days during co-culture with ECs. P values are shown for comparisons between monoculture and 1:1 ratio. Representative flow cytometry histograms and mean fluorescence intensity (MFI) quantification for day 7 of co-culture are shown. e , Gating strategy for classifying COMP hi and POSTN hi fibroblasts from the Xenium-profiled co-culture (mutually exclusive top quantile of cells expressing each gene, with POSTN ≤ 3.5 for COMP hi ). f , Representative examples of COMP hi and POSTN hi fibroblasts in 2D culture are shown with transcripts. g , Violin plot showing the distribution of TGFBR2 and TGFBR3 transcripts on POSTN hi and COMP hi gated fibroblasts in 2D co-culture, 3D co-culture and RA synovium. h , Representative example ( n = 4 RA synovial tissue) of gated (mutually exclusive top quantiles of expressing cells) COMP hi and POSTN hi synovial fibroblasts with transcripts. i , j , ELISA quantification of COMP ( i ) and POSTN ( j ) production from co-culture of fibroblasts overexpressing (OE) GFP , TGFBR2 or TGFBR3 with ECs in a 1:1 ratio. P values are shown for the comparison between TGFBR3 -OE and GFP -OE data points. The bar plot on the right represents the area under the curve. Data points are shown as the mean ± s.d., represent n = 3 biological replicates and are representative of at least two independent experiments. For statistical analysis, a two-tailed Student’s t -test was used for a – d , i and j , and a two-sided Wilcoxon test was used for g .

Article Snippet: Fibroblasts or co-cultures were trypsinized and directly stained with an equal volume of primary antibody solution (Cell staining buffer, BioLegend) containing eFluor 780 Fixable Viability Dye (Thermo Fisher Scientific; 1:1,000 dilution), TGFβR2-APC (BioLegend; 1:100 dilution), TGFβRIII (Proteintech, 20000-1-AP; 1:100 dilution) or CD31-FITC (BioLegend; 1:100 dilution) for 30 min at 4 C. Cells were washed with cell staining buffer (BioLegend), fixed in 4% paraformaldehyde for 10 min at 20 °C, washed, and stained with AF555 anti-rabbit (Thermo Fisher Scientific) for 30 min at 4 °C for detection of TGFβIII.

Techniques: Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Flow Cytometry, Fluorescence, Expressing, Comparison, Two Tailed Test