recombinant human tgf beta 1 protein tgfβ1  (R&D Systems)

Journal: Cell Death Discovery

Article Title: WWP1 gain-of-function drives developmental anoikis through TGFβ pathway during neurodevelopment

doi: 10.1038/s41420-026-02977-4

Figure Lengend Snippet: A Scheme of WWP1 variant-expressing cell line establishment and downstream analysis. The image is created with Biorender.com. B Representative images of dead cell population in WWP1 variants-expressing HeLa cell lines treated with gefitinib. Red, propidium iodide (PI). Scale bar, 200 μm. C Quantification of the PI + counts in HeLa cell lines treated with gefitinib. n = 3 for each condition. Two-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01. D Functional GO enrichment analysis of upregulated and downregulated DEGs between WWP1 E798V and control cell lines. The bar color indicates the cluster categories of the GO term. BP, biological process; CC, cellular component. E Differential PROGENy pathway activity scores of WWP1 E798V relative to control. The pathway with the lowest value, TGFβ, is indicated by a deep blue color. F Gene set enrichment plots for TGFβ signaling and epithelial-mesenchymal transition. NES, normalized enrichment score. G Western blot analysis of phospho-SMAD2 and SMAD2 in WWP1 variant-expressing HeLa cell lines treated with vehicle or hTGFβ1 (10 ng/mL). ACTIN is used as the loading control. H Quantification of normalized pSMAD2 to SMAD2 protein expression ratio in WWP1 variant-expressing HeLa cell lines. n = 5. Kruskal-Wallis test with Dunn’s post hoc test. * p < 0.05; ns not significant. Bar graphs indicate mean ± SEM.

Article Snippet: Z-VAD-FMK (20 μM, MedChemExpress, # HY-16658B), recombinant human TGFβ1 (5 μg/mL), or galunisertib (1 or 5 μM, MedChemExpress, #HY-13226) were administered with plasmids intracerebroventricularly during IUE.

Techniques: Variant Assay, Expressing, Functional Assay, Control, Activity Assay, Western Blot

Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: Chol-Loading Downregulates TGFβ Signaling in hVSMC hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin (CT; ie, 0 μg/mL cholesterol) for 24 hours in the presence or absence of TGFβ1 ligand (10 pg/mL). Total RNA was isolated and quantitative polymerase chain reaction (qPCR) was performed to determine the pri-Mir143/145 precursor transcripts (A,B) or SMC markers, Acta2 and Tagln (C,D). hVSMCs were treated as in A and B, but either in the presence or absence of TGFβ1 10 pg/mL) and/or nonscrambled (NS) or Mir145 mimic (60 nmol/L). qPCR was performed to determine expression of Acta2 (E) and (F) Srf mRNA. (G) hVSMCs were treated as in A and B, but either in the presence or in absence of TGFβ1 (10 pg/mL) and/or Mir145 inhibitor (60 nmol/L). qPCR was performed to determine expression of Acta2. (H) Immunofluorescence images of total SMAD2/3 (green) in hVSMCs after 24 hours of the indicated treatments. Cytoplasm was stained with phalloidin (red). Nuclei were determined as phalloidin negative area (bar = 50 μm). (I) hVSMCs were treated as in A and B, but with varying amounts of Chol and in the presence or absence of recombinant TGFβ1 (10 pg/mL) for 24 hours. Proteins were extracted for Western blotting to detect phosphorylated (p) SMAD2/3, and α-SMA. Total SMAD2/3 or GAPDH was used as loading CT proteins. Blots are representative of at least 3 independent experiments, and the replicates were quantified by densitometry. For data comparisons of 2 or more independent groups, 1-way or 2-way analysis of variance followed by Dunnett post hoc test was performed. Data are presented as the mean ± SEM of 3 independent experiments, and P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). ns = not significant; other abbreviations as in .

Article Snippet: For cholesterol or TGFβ1 treatment, cells were serum starved for 24 hours in 0.2% bovine serum albumin (BSA) (in basal media without serum; #310-500, Cell Applications), and treatments including methyl-β-cyclodextrin-cholesterol mixture (5 μg/mL, Sigma; hereafter referred to as cholesterol treatment), methyl-β-cyclodextrin (20 mmol/L; Sigma) TGFβR1 inhibitor (SB431542, Sigma), and recombinant human TGFβ1 (R&D Systems) were performed.

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Staining, Recombinant, Western Blot

Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: Cholesterol-Loading Partitions TGFβ Receptors Into Membrane Lipid Rafts hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin for 24 hours. (A) Membrane lipid raft (LR) and nonraft (NR) fractions were isolated, and Western blotting was performed using each of these fractions to determine the expressions of TGFβR1 and TGFβR2, as well as CAV1, flotillin, and transferrin receptor (CD71). (B) Densitometry was performed to quantify the levels of TGFβR1 and TGFβR2 in the LR and NR fractions. (C) Western blotting was performed from total cell lysates of Chol-treated or untreated cells, and the bands of the TGFβ receptors visualized. (D and E) Densitometry was performed to quantify the levels of TGFβR1 and TGFβR2. Blots are representative of 3 independent experiments. For data analysis, unpaired Student’s t -test was performed for comparing the means of 2 groups. For 2 or more independent groups, 2-way analysis of variance followed by Dunnett post hoc test was performed. Data are presented as the mean ± SEM of at least 3 independent experiments, and P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01). Abbreviations as in .

Article Snippet: For cholesterol or TGFβ1 treatment, cells were serum starved for 24 hours in 0.2% bovine serum albumin (BSA) (in basal media without serum; #310-500, Cell Applications), and treatments including methyl-β-cyclodextrin-cholesterol mixture (5 μg/mL, Sigma; hereafter referred to as cholesterol treatment), methyl-β-cyclodextrin (20 mmol/L; Sigma) TGFβR1 inhibitor (SB431542, Sigma), and recombinant human TGFβ1 (R&D Systems) were performed.

Techniques: Membrane, Isolation, Western Blot

Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: HDL Treatment In Vitro Restores TGFβ Signaling in Cholesterol-Loaded hVSMCs (A) hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin for 24 hours, followed by high-density lipoprotein (HDL) (50 μg/mL) treatment for 48 hours. Then, treatment groups were stimulated with recombinant TGFβ1 (10 pg/mL). Western blotting was performed to detect pSMAD2 and total (t) SMAD2, with densitometry used for quantification. (B to E) qPCR was performed to detect expression of Mir143/145, Myocd, Acta2, Cnn1, and Hmgcr at the conclusion of the experiment in A. (F) Chol-loaded cells were either treated with HDL alone, HDL + TGFβR1 antagonist (TGFβR1i; 50 ng/mL), or left untreated. Western blotting was performed to detect α-SMA. GAPDH was used as loading CT protein. For data analysis of 2 or more independent groups, 1-way or 2-way analysis of variance followed by Dunnett post hoc test was performed. Blots are representative of 3-5 independent experiments (mean ± SEM). P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). Abbreviations as in and .

Article Snippet: For cholesterol or TGFβ1 treatment, cells were serum starved for 24 hours in 0.2% bovine serum albumin (BSA) (in basal media without serum; #310-500, Cell Applications), and treatments including methyl-β-cyclodextrin-cholesterol mixture (5 μg/mL, Sigma; hereafter referred to as cholesterol treatment), methyl-β-cyclodextrin (20 mmol/L; Sigma) TGFβR1 inhibitor (SB431542, Sigma), and recombinant human TGFβ1 (R&D Systems) were performed.

Techniques: In Vitro, Recombinant, Western Blot, Expressing

Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: HDL Treatment Displaces TGFβ Receptor From Membrane Lipid Rafts in Chol-Loaded hVSMCs and Restores its Signaling hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin (CT) for 24 hours, after which they were all treated with HDL (50 μg/mL) for 24 hours. (A) At the end of the 48-hour protocol, LR and NR fractions were isolated, and Western blotting was performed using each of these fractions to determine the expressions of TGFβR1 and TGFβR2, as well as CAV1, and flotillin. Densitometry was performed to quantify the level of (B) TGFβR1 and (C) TGFβR2. (D) hVSMCs were loaded with Chol (48 hours, 5 μg/mL) and were then either treated with HDL (50 μg/mL) for 24 hours, or left untreated. Western blotting was performed to determine pSMAD2, SMAD2, and GAPDH levels. For data analysis of 2 or more independent groups, 2-way analysis of variance followed by Šídák multiple comparisons post hoc test was performed. Data are presented as the mean ± SEM of at least 3 independent experiments, and the P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01). Abbreviations as in , , , .

Article Snippet: For cholesterol or TGFβ1 treatment, cells were serum starved for 24 hours in 0.2% bovine serum albumin (BSA) (in basal media without serum; #310-500, Cell Applications), and treatments including methyl-β-cyclodextrin-cholesterol mixture (5 μg/mL, Sigma; hereafter referred to as cholesterol treatment), methyl-β-cyclodextrin (20 mmol/L; Sigma) TGFβR1 inhibitor (SB431542, Sigma), and recombinant human TGFβ1 (R&D Systems) were performed.

Techniques: Membrane, Isolation, Western Blot

Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: Macrophage Markers Upregulated in Chol-Loaded hVSMCs Are Suppressed by HDL Through Restoration of TGFβ Signaling (A) hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin (CT) for 48 hours. qPCR was performed to determine the expression of macrophage marker ( Cd68 ) and SMC marker ( Acta2 ). (B) hVSMCs were with treated as in A for 48 hours, then qPCR was performed to determine the expression of macrophage differentiation factor Klf4 . (C) hVSMCs were treated with Chol (5 μg/mL) for the indicated times, then KFL4 expression was determined by Western blotting. (D) Klf4 (60 nmol/L) or negative CT small, interfering RNA (siRNA) were transfected into hVSMCs for 48 hours. Then, transfected cells were treated as in B, followed by Western blotting for CD68 and KLF4. GAPDH was used as loading CT. (E) Chol-loaded cells (48 hours, 5 μg/mL) were incubated with Mir145 mimic (60 nmol/L) or CT mimic (60 nmol/LM) for 24 hours and the expressions of CD68, KLF4, and α-SMA determined with GAPDH as a loading CT. The P values for the comparisons between CT and Mir145 mimics are CD68 (0.025), KLF4 (0.018), and α-SMA (0.01). (F-I) hVSMCs were loaded with Chol (48 hours, 5 μg/mL) and were then either treated with HDL (50 μg/mL) for 24 hours or left untreated. Western blotting was performed to determine the expression of (F) KLF4 and (G) CD68. (H) hVSMCs were treated as in F and G, but in the presence or absence of TGFβR1i (50 ng/mL). Western blotting was performed to determine KLF4 expression. For data analysis, unpaired Student’s t -test was performed for comparing the means of 2 groups. For 2 or more independent groups, 2-way analysis of variance followed by Dunnett post hoc test was performed. Data are presented as the mean ± SEM of at least 3 independent experiments. P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). Abbreviations as in , , and .

Article Snippet: For cholesterol or TGFβ1 treatment, cells were serum starved for 24 hours in 0.2% bovine serum albumin (BSA) (in basal media without serum; #310-500, Cell Applications), and treatments including methyl-β-cyclodextrin-cholesterol mixture (5 μg/mL, Sigma; hereafter referred to as cholesterol treatment), methyl-β-cyclodextrin (20 mmol/L; Sigma) TGFβR1 inhibitor (SB431542, Sigma), and recombinant human TGFβ1 (R&D Systems) were performed.

Techniques: Expressing, Marker, Western Blot, Small Interfering RNA, Transfection, Incubation

Journal: bioRxiv

Article Title: WISP1 drives a mechanically active immune modulatory and proliferative cardiac myofibroblast state

doi: 10.64898/2026.02.17.706476

Figure Lengend Snippet: (A) Wisp1 mRNA expression in primary adult mouse cardiac fibroblasts following TGFβ1 (10 ng/mL) treatment for 24–72 h. (B) Representative immunofluorescence images of α-SMA (green), vimentin (red), and DAPI (blue) after 72 h treatment with vehicle (Ctrl), WISP1 (500 ng/mL), TGFβ1 (10 ng/mL), or WISP1 + TGFβ1. (C, D) Representative Western blot (C) and densitometric quantification (D) of α-SMA normalized to GAPDH. Data are mean ± SEM from ≥3 independent isolations. Statistical analysis by one-way ANOVA with Tukey’s post hoc test; *p < 0.05. Data are mean ± SEM from ≥3 independent isolations. Statistical analysis by one-way ANOVA with Tukey’s post hoc test; *p < 0.05.

Article Snippet: For treatments, CFs were seeded in 12- or 24-well plates in DMEM + 10% FBS and treated for 72 h with recombinant human TGFβ1 (10 ng/mL; R&D Systems, 7754-BH) and/or recombinant murine WISP1 (500 ng/mL; R&D Systems, 1680-WS-050).

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: WISP1 drives a mechanically active immune modulatory and proliferative cardiac myofibroblast state

doi: 10.64898/2026.02.17.706476

Figure Lengend Snippet: Primary adult mouse cardiac fibroblasts were embedded in floating collagen gels and treated for 24 h with vehicle (Ctrl), WISP1 (500 ng/mL), TGFβ1 (10 ng/mL), or WISP1 + TGFβ1. (A) Representative gel images at 0 h and 24 h. (B) Quantification of gel area (% contraction; n = 4 isolations, 2 males & 2 females). (C–D) Confluent fibroblast monolayers were scratched and imaged every 2 hours for 24 hours using the same treatment conditions. (C) Representative bright-field images at time point 0 and 14. (D) Quantification of wound closure (% of gap closed relative to time 0; n = 8 isolations, 4 male & 4 female). Data are mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons; *p ≤ 0.05.

Article Snippet: For treatments, CFs were seeded in 12- or 24-well plates in DMEM + 10% FBS and treated for 72 h with recombinant human TGFβ1 (10 ng/mL; R&D Systems, 7754-BH) and/or recombinant murine WISP1 (500 ng/mL; R&D Systems, 1680-WS-050).

Techniques:

Journal: Advanced Science

Article Title: TGFβ1 Activates Lnc‐APUE to Promote Tumor Metastasis via the Alu Element‐Driven STAU1‐Mediated Decay of CDH1 mRNA

doi: 10.1002/advs.202518731

Figure Lengend Snippet: TGFβ1 activates lnc‐APUE transcription. (A) Schematic diagram of Firefly luciferase reporters containing the specific DNA fragments of the lnc‐APUE promoter. Arrow denotes the transcription start site (TSS) and transcription direction of lnc‐APUE. Short vertical line, putative SMAD‐binding element (SBE). Red triangle (Δ), deletion of the SBE (delSBE). Red diamond (◊), mutation of the SBE (mutSBE). (B) TGFβ1 treatment enhanced the activity of the lnc‐APUE promoter. (C) The −0.5 to −0.2‐kb region of the lnc‐APUE promoter contained TGFβ1 responsive elements. (D) Deletion of putative SBE1 and SBE2 in the lnc‐APUE promoter abrogated the response of P(−0.5/+0.07k) reporter to TGFβ1. For (B–D), HCCLM9 cells were transfected with the indicated vectors for 36 h. The cells were then either left untreated or treated with TGFβ1 for a further 12 h prior to the luciferase activity assay. (E,F) ChIP analysis showed a direct interaction between SMAD2 and the lnc‐APUE promoter in vivo. ChIP analysis was performed in HCCLM9 cells using anti‐SMAD2 or isotype‐matched IgG, and the antibody‐precipitated DNAs were examined by semi‐quantitative PCR assay (E) or by qPCR analysis (F). Lnc‐APUE promoter amplicons spanning the regions −1537 to −1405‐bp, −969 to −844‐bp, −528 to −424‐bp, and −159 to −39‐bp, were analyzed. The CDH2 and GAPDH promoters were included as positive and negative controls, respectively. The data from at least three independent experiments are presented as mean ± SEM (B–D,F); p values were assessed by unpaired Student′s t ‐test (B–D, F). * , p < 0.05; ** , p < 0.01; *** , p < 0.001; ns, not significant.

Article Snippet: Reagents were purchased as follows: TGFβ1 (240‐B‐002, R&D Systems, Minneapolis, MN, USA); SB525334 (S1476, Selleckchem, Houston, TX, USA); actinomycin‐D (ActD, 15021 S, Cell Signaling Technology, CST, Beverly, MA, USA).

Techniques: Luciferase, Binding Assay, Mutagenesis, Activity Assay, Transfection, In Vivo, Real-time Polymerase Chain Reaction

Journal: Advanced Science

Article Title: TGFβ1 Activates Lnc‐APUE to Promote Tumor Metastasis via the Alu Element‐Driven STAU1‐Mediated Decay of CDH1 mRNA

doi: 10.1002/advs.202518731

Figure Lengend Snippet: TGFβ1/SMAD signaling promotes tumor metastasis by enhancing lnc‐APUE expression. (A,B) TGFβ1 increased lnc‐APUE levels in hepatoma cells in a time‐ and dose‐dependent manner. HCCLM9 and Huh‐7 cells were untreated or treated with 1 ng/mL TGFβ1 for the indicated time (A) or with the indicated dose of TGFβ1 for 24 h (B). (C–E) Inhibition of TGFBR1 or simultaneous silencing of SMAD2/3/4 abolished the promotive role of TGFβ1 in lnc‐APUE expression. For (C), cells were untreated (‐) or treated with (+) TGFβ1 or TGFBR1 inhibitor (SB525334) for 12 h. For (D,E), HCCLM9 and Huh‐7 were transfected with the indicated RNA duplexes and then incubated without (‐) or with (+) TGFβ1 for 24 h before qPCR analysis. (F) Silencing lnc‐APUE abrogated the effects of TGFβ1 in reducing E‐cadherin levels and in promoting migration and invasion. HCCLM9 transfected with the indicated siRNAs were incubated without (‐) or with (+) TGFβ1 for 36 h, then subjected to western blotting ( Top panel), migration ( middle panel), and invasion ( bottom panel) assays. Lnc‐APUE is abbreviated as “APUE”. RNAiMAX, cells exposed to Lipofectamine RNAiMAX without RNA. NC, negative control for siRNA. Data from at least three independent experiments are presented as mean ± SEM (A–F); p values were assessed by one‐way ANOVA (A–F). * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, not significant.

Article Snippet: Reagents were purchased as follows: TGFβ1 (240‐B‐002, R&D Systems, Minneapolis, MN, USA); SB525334 (S1476, Selleckchem, Houston, TX, USA); actinomycin‐D (ActD, 15021 S, Cell Signaling Technology, CST, Beverly, MA, USA).

Techniques: Expressing, Inhibition, Transfection, Incubation, Migration, Western Blot, Negative Control

Journal: Advanced Science

Article Title: TGFβ1 Activates Lnc‐APUE to Promote Tumor Metastasis via the Alu Element‐Driven STAU1‐Mediated Decay of CDH1 mRNA

doi: 10.1002/advs.202518731

Figure Lengend Snippet: Schematic illustration of the TGFβ1/SMAD/lnc‐APUE/E‐cadherin axis and its regulatory role in tumor metastasis (By Figdraw).

Article Snippet: Reagents were purchased as follows: TGFβ1 (240‐B‐002, R&D Systems, Minneapolis, MN, USA); SB525334 (S1476, Selleckchem, Houston, TX, USA); actinomycin‐D (ActD, 15021 S, Cell Signaling Technology, CST, Beverly, MA, USA).

Techniques: