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: Biomedicines

Article Title: Derazantinib Inhibits the Bioactivity of Keloid Fibroblasts via FGFR Signaling.

doi: 10.3390/biomedicines11123220

Figure Lengend Snippet: Figure 3. KFs were treated with 2.5 µmol derazantinib or without derazantinib for 48 h. (A) The PCR showed that the expression of fibrotic genes was suppressed in the experiment group. (B–D) The immunofluorescence staining showed that collagen I, α-SMA, and PAI-1 expression were decreased in the derazantinib group. The length of the scale is 50 µm. (E,F) The WB showed that the protein production of a-SMA, collagen I, PAI-1, and FGFR1 in the derazantinib group was suppressed. (G) The PCR showed that the expression of PI3K and JNK genes was suppressed in the experiment group. (H) The WB showed that the expression of ERK, p-ERK, AKT, TGF-β, p-AKT, and PI3K in the derazantinib group was suppressed. No significant effect was found on the SMAD expression levels. * p < 0.05 compared to control group.

Article Snippet: The PVDF membrane was separately treated with the specific primary antibodies: α-SMA (AF1032, Affinity, Changzhou, China, rabbit), collagen I (66948, CST, USA, mouse), PAI-1 (13801-1-AP, Proteintech, IL, USA, rabbit), FGFR1 (60325-1-Ig, Proteintech, USA, mouse), GAPDH (GB11002, Servicebio, Wuhan, China, rabbit), PI3K (AF5112, Affinity, Changzhou, China, rabbit), p-AKT (AF0016, Affinity, Changzhou, China, rabbit), AKT (BS0115R, Bioss, Wuhan, China, rabbit), p-ERK (AF1015, Affinity, Changzhou, China, rabbit), ERK (AF0155, Affinity, Changzhou, China, rabbit), TGF-β (BA0290, Bosterbio, Beijing, China, rabbit), Smad (6367, Affinity, Changzhou, China, rabbit), and p-Smad (3367, Affinity, Changzhou, China, rabbit).

Techniques: Expressing, Staining, Control

Journal: Frontiers in Immunology

Article Title: DNA Damage Response Evaluation Provides Novel Insights for Personalized Immunotherapy in Glioma

doi: 10.3389/fimmu.2022.875648

Figure Lengend Snippet: DDR-associated immune microenvironment signatures and characteristics. (A) The differentially expressed TME signatures in high and low DDR score groups. (B) The expression levels of immune checkpoints in high and low DDR score groups. (C) The expression levels of cGAS-STING pathway members in high and low DDR score groups. (D) IHC staining of PD-L1 and TGFβ in primary and recurrent glioma tissues (20×magnification). EMT, epithelial-to-mesenchymal transition; MHC, major histocompatibility complex; HLA, human leukocyte antigen; IFN, interferon; Pan-F-TBR, pan-fibroblast TGFβ response. ****p < 0.0001.

Article Snippet: The samples were incubated with primary anti-PD-L1 (1:200, ab237726, Abcam, Cambridge, UK) or anti-TGFβ antibody (1:200, BA0290, BOSTER, Wuhan, China) overnight at 4°C.

Techniques: Expressing, Immunohistochemistry, Immunopeptidomics