Journal: Materials today. Bio

Article Title: Mi-BMSCs alleviate inflammation and fibrosis in CCl 4 -and TAA-induced liver cirrhosis by inhibiting TGF-β/Smad signaling.

doi: 10.1016/j.mtbio.2024.100958

Figure Lengend Snippet: Fig. 1. Schematic illustration of BMSCs-loaded porous particles fabrication. a. The generation of porous microcapsules with microfluidic electrospray; b. BMSCs- encapsulated microspheres intraperitoneally regulate the inflammatory microenvironment to alleviate liver function and fibrosis through paracrine and TGF- β/Smad signaling pathway for liver cirrhosis.

Article Snippet: PCNA, IL-6, N-Cadherin, E-Cadherin, and TGF-β Fibrosis Pathway Antibody Sampler Kit antibodies were obtained from CST (USA).

Techniques:

Journal: Materials today. Bio

Article Title: Mi-BMSCs alleviate inflammation and fibrosis in CCl 4 -and TAA-induced liver cirrhosis by inhibiting TGF-β/Smad signaling.

doi: 10.1016/j.mtbio.2024.100958

Figure Lengend Snippet: Fig. 4. Therapeutic efficacy of Mi-BMSCs or BMSCs in TGF-β1-induced HSCs activation. (a) Cell viability of inactivated HSCs with different treatments at 24h, 48 h. (b–c) Total apoptosis of activated HSCs with different treatments at 24 h assayed by flow cytometry analysis (upper and lower right quadrants represent late and early apoptotic cells). (d–f) The relative mRNA levels of α-SMA, TGF-β, and IL-6 measured by qRT-PCR in Control, TGF-β1, BMSCs, Mi-BMSCs group. (g–l) Western blot and quantification analysis of COL1A1 (h), E-cadherin (i), N-cadherin (j), TGF-β R II (k) and TGF-β (l) in Control, TGF-β1, BMSCs, Mi-BMSCs group. Data are expressed as Means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs TGF-β1 group.

Article Snippet: PCNA, IL-6, N-Cadherin, E-Cadherin, and TGF-β Fibrosis Pathway Antibody Sampler Kit antibodies were obtained from CST (USA).

Techniques: Drug discovery, Activation Assay, Flow Cytometry, Quantitative RT-PCR, Control, Western Blot

Journal: Materials today. Bio

Article Title: Mi-BMSCs alleviate inflammation and fibrosis in CCl 4 -and TAA-induced liver cirrhosis by inhibiting TGF-β/Smad signaling.

doi: 10.1016/j.mtbio.2024.100958

Figure Lengend Snippet: Fig. 10. The mechanism of Mi-BMSCs in the treatment of liver cirrhosis. (a–b) The representative fluorescent images and quantification of TGF-β from liver tissues of mice in Control, LF, BMSCs, Mi-BMSCs group by Immunofluorescent (IF) staining. Scale bar = 100 μm. (c) The serum levels of TGF-β in each group by ELISA. (d) The mRNA levels of TGF-β from liver tissues in each group by qRT-PCR assay. (e–h) The protein relative expression and quantification of TGF-β (g), the phosphorylation of Smad2 (f) and Smad2/3 (g) of liver tissues in Control, LF, BMSCs, Mi-BMSCs group by WB analysis *p < 0.05, **p < 0.01, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs LF group; Data are expressed as Means ± SD.

Article Snippet: PCNA, IL-6, N-Cadherin, E-Cadherin, and TGF-β Fibrosis Pathway Antibody Sampler Kit antibodies were obtained from CST (USA).

Techniques: Control, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Phospho-proteomics

Journal: Advanced Science

Article Title: A Multifunctional Therapeutic Strategy Using P7C3 as A Countermeasure Against Bone Loss and Fragility in An Ovariectomized Rat Model of Postmenopausal Osteoporosis

doi: 10.1002/advs.202308698

Figure Lengend Snippet: P7C3 is non‐cytotoxic and promotes hBMSC osteogenesis while inhibiting adipogenesis. A] The MTT assay was performed to determine cell metabolic activity. hBMSCs were treated with 0, 1 or 10 µM of P7C3 for 3 days and 5 days. 1 or 10 µM of DMSO were used as a vehicle control. B] Representative confocal microscopy images of hBMSCs treated with or without 10 µM of P7C3. The cells were fixed, stained with phalloidin (red) and DAPI (blue), and examined using confocal laser scanning microscopy. C] ALP staining results of hBMSCs cultured in osteogenic induction medium supplemented with P7C3 or DMSO vehicle (day 7). Representative ALP‐stained micrographs are shown in the lower panels of each figure. D] Alizarin red S staining for mineral deposition. Mineral deposition appears bright red in color (day 14). E] Quantification of mineralized calcium nodules (day 14). F] Alizarin red S staining results (day 28). Intense mineralized calcium nodule staining was noted following 10 µM P7C3 treatment. G] Quantification of mineralized calcium nodules (day 28). H] Results from a human bone metabolism array and quantification via a heat map. hBMSCs were either treated with 10 µM DMSO or 10 µM P7C3 in osteogenic differentiation media for 21 days. The cells were collected for array analysis. I] A human TGF‐β pathway phosphorylation array and quantification via a heat map. hBMSCs were either treated with 10 µM DMSO or 10 µM P7C3 in osteogenic differentiation media for 1 h. The cells were collected for array analysis. J–L] Representative phase contrast and LipidSpot™ Lipid Droplet‐stained images of hBMSCs cultured in adipogenic induction medium supplemented with P7C3 or the DMSO vehicle for 14 days J] and 21 days L]. The quantification of LipidSpot™ + cells is presented in image K. M] Heat map of mRNA expression levels of adipogenesis‐related genes were measured after 12 days of culture. hBMSCs were cultured in adipogenic induction medium supplemented with either P7C3 or DMSO for 12 days. The gene expression of ADPOQ , SREBP‐1 , CEBPA , FABP4 , and LPL were investigated. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The human bone metabolism array (AAH‐TGFB‐1‐2, Raybiotech, USA) was performed following the manufacturer's instructions.

Techniques: MTT Assay, Activity Assay, Control, Confocal Microscopy, Staining, Confocal Laser Scanning Microscopy, Cell Culture, Phospho-proteomics, Expressing, Gene Expression

Journal: Journal of Neurogenetics

Article Title: Molecular Profiles of Pyramidal Neurons in the Superior Temporal Cortex in Schizophrenia

doi: 10.3109/01677063.2014.882918

Figure Lengend Snippet: Overrepressented KEGG pathways based on enrichment analysis of the predicted target genes of the differentially expressed miRNAs.

Article Snippet: We found that the TGF-β and BMP signaling canonical pathways were highly dysregulated in schizophrenia ( GeneGo : p < 0.0001), with up-regulation of several genes immediately downstream from the TGF-β1 receptor ( available online at http://informahealthcare. com/doi/abs/10.3109/01677063.2014.882918 ; ).

Techniques:

Journal: Scientific Reports

Article Title: TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT

doi: 10.1038/srep11924

Figure Lengend Snippet: A : Summary of altered expression of lncRNAs in the CAF-CM-treated 5637 and J82 cells, comparing to the NF-CM-treated ones. 1.5-fold of relative mRNA level using qRT-PCR, normalized by β-actin gene, was used as the threshold for significant changes. B and C : ZEB2NAT expression levels in the CAF-CM treated 5637 and J82 cells upon the treatments of a TGFβ1 neutralizing antibody ( B ) and a TGFβRI inhibitor (SB-431542) ( C ), respectively. D : Exogenous expression of ZEB2NAT lncRNA in 5637 cells, detected by qRT-PCR using β-actin gene as the normalization control. E – G : Effects of ZEB2NAT lncRNAs overexpression on cell invasion by the Transwell invasion assay ( E , F ) and protein levels of E-cadherin, ZEB1 and ZEB2 by immunoblotting ( G ) in 5637 cells. H: Knockdown of ZEB2NAT by RNAi using two siRNAs targeting different regions of the lncRNA (siZEB2NAT-1 and siZEB2NAT-2) in 5637 cells. The relative ZEB2NAT expression levels were at 48 hours after siRNA transfection and determined by qRT-PCR using β-actin gene as the normalization control. I , J , K : Effects of ZEB2NAT lncRNAs knockdown on cell invasion by the Transwell invasion assay ( I , J ) and protein levels of E-cadherin and ZEB2 by immunoblotting ( K ) in the CAF-CM treated 5637 cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: As for lncRNA profiling, we used Human lncRNA Discover PCR array/TGFβ pathway (Bio-Serve Company, BS-lncRNA002, Shanghai, China), which consisting of 72 lncRNAs, derived from the lncRNA database ( www.lncRNAdb.org ) and several positive genes which are regulated by TGFβ, such as Vimentin.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Transwell Invasion Assay, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Impairment of Transforming Growth Factor ? Signaling in Caveolin-1-deficient Hepatocytes

doi: 10.1074/jbc.M109.072900

Figure Lengend Snippet: Cav-1 and TGF-β-signaling molecule expression in regenerating liver after PH. Total tissue extracts were prepared from Cav-1+/+ and Cav-1−/− remnant liver after PH at different times. A, protein levels of TβR-I and -II, total and pSmad2/3, Cav-1, SnoN, and PAI-1 were determined by Western blot. Representative blots are shown. B, densitometric analysis of the immunoblots. Data are presented as the means ± S.D. of five independent experiments (animals). *, p < 0.01 versus the corresponding value at 0 time. a, p < 0.01 versus the corresponding value at the indicated time in Cav-1+/+ mice. Blots were normalized with p85 for total extracts.

Article Snippet: The mouse TGF-β/BMP signaling pathway PCR array was performed according to the manufacturer's protocol, using the Profiler PCR array system and the SYBR Green/fluorescein qPCR master mix (SuperArray Bioscience) on a MyiQ real time PCR system (Bio-Rad).

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Impairment of Transforming Growth Factor ? Signaling in Caveolin-1-deficient Hepatocytes

doi: 10.1074/jbc.M109.072900

Figure Lengend Snippet: TβR-I/TβR-II interaction is decreased in the absence of Cav-1. A, protein levels of Cav-1 in NCL lines after treatment with 5 ng/ml TGF-β1 for 30 min. B, time-dependent proliferation of NCL Cav-1+/+ and NCL Cav-1−/− cells. Cells were seeded in medium containing 10% (v/v) FBS and, after the indicated times in culture, were trypsinized, stained with trypan blue, and counted. a.u., arbitrary units. C, sucrose discontinuous (5–30-40%) gradients were performed using a Triton X-100-based method to isolate DRM or NDRM fractions from the NCL cells as described under “Experimental Procedures.” Cav-1 is mainly localized in the fractions 4–5 in NCL Cav-1+/+ cells. GM130 (GA), calregulin (ER), annexin VI (RREs), and 5′-nucleotides (5′NU) (DRM) were used as subcellular markers. D, Western blot detection of TβR-I and II, Smad2/3, and Cav-1 in 50 μl from fractions 4 to 5 for DRM and fractions 10 to 11 for NDRM from NCL cells. Whole cell lysates (WCL) (50 μg) were used as reference for total content and blotted with the same Abs. E, quantification (%) of the distribution of the corresponding protein analyzed in D. The results are expressed as the band intensity of the protein in DRM (fractions 4 to 5) and NDRM (fractions 10 to 11) fractions with respect to the total protein levels. Representative blots are shown. Results are the means ± S.D. of four independent experiments. *, p < 0.01 versus the corresponding value at 0 time (B) or the corresponding value in NCL Cav-1+/+ (E). a, p < 0.01 versus the corresponding value at the indicated time in NCL Cav-1+/+. Blots were normalized with p85 for total extracts.

Article Snippet: The mouse TGF-β/BMP signaling pathway PCR array was performed according to the manufacturer's protocol, using the Profiler PCR array system and the SYBR Green/fluorescein qPCR master mix (SuperArray Bioscience) on a MyiQ real time PCR system (Bio-Rad).

Techniques: Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Impairment of Transforming Growth Factor ? Signaling in Caveolin-1-deficient Hepatocytes

doi: 10.1074/jbc.M109.072900

Figure Lengend Snippet: SnoN accumulates in NCL Cav-1−/− cells. NCL Cav-1+/+ and NCL Cav-1−/− cells (2–3 × 106) were treated with 5 ng/ml TGF-β1 at the indicated times. Cytosolic (50 μg) (A) and nuclear extracts (10 μg) (B) were obtained, and protein levels of total and pSmad2/3, PAI-1, and SnoN were analyzed. A representative blot is shown. Blots were normalized with p85 for cytosolic extracts and with histone-H1 for nuclear extracts. C, immunofluorescence analysis of SnoN in NCL cells after 1 h of TGF-β1 stimulation. DAPI, 4′,6-diamidino-2-phenylindole. D, immunofluorescence analysis of pSmad2/3 in NCL cells after TGF-β1 stimulation at the indicated times. Neonatal Cav-1+/+ or Cav-1−/− cells (5 × 104) were cultured in 24-multiwell plates on glass coverslips and maintained overnight with 1% FBS. After that period, the cells were fixed for 15 min with 4% paraformaldehyde, pH 7.2, washed with phosphate-buffered saline, and permeabilized with 1% Tween 20 in phosphate-buffered saline for 15 min at room temperature. After blocking with 3% bovine serum albumin for 1 h at room temperature, the cells were incubated 2 h with the corresponding Abs diluted 1:150 in 1% bovine serum albumin, washed several times, and incubated for 2 h with fluorochrome-conjugated Abs (Invitrogen) raised against Fc of primary Abs and treated with 4′,6-diamidino-2-phenylindole for 30 min at room temperature. The glass coverslips were mounting with Vectashield (Vector Laboratories, Burlingame, CA) on microscope slides. The images were acquired in a fluorescence Eclipse E400 microscope (Nikon).

Article Snippet: The mouse TGF-β/BMP signaling pathway PCR array was performed according to the manufacturer's protocol, using the Profiler PCR array system and the SYBR Green/fluorescein qPCR master mix (SuperArray Bioscience) on a MyiQ real time PCR system (Bio-Rad).

Techniques: Immunofluorescence, Cell Culture, Blocking Assay, Incubation, Plasmid Preparation, Microscopy, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Impairment of Transforming Growth Factor ? Signaling in Caveolin-1-deficient Hepatocytes

doi: 10.1074/jbc.M109.072900

Figure Lengend Snippet: TGF-β-responsive promoter and PAI-1 expression are impaired in the absence of Cav-1. A, NCL Cav-1+/+ and NCL Cav-1−/− cells were transfected with pEGFN1 (encoding wild-type Cav-1-GFP or GFP) and with the 3TP-Lux vectors with FuGENE-6 (Roche Applied Science) for 12 h. After 24 h of transfection, cells were treated with TGF-β1 for 12 h, and the luciferase (Luc.) activity was measured by using the luciferase assay system. Inset, SnoN expression in NCL cells after transfection with Cav-1-GFP vector. a.u., arbitrary units. B, Western blot (WB) analysis of the protein, Cav-1-GFP, GFP, or Cav-1, expressed after transfection with the pEGFPN1 vector as a control of the process (C, Cav-1; G, GFP). C, 1 μg of total RNA, extracted with TRIzol reagent (Invitrogen), was reverse-transcribed using SuperScriptTM III first-strand synthesis system for RT-PCR. Real time PCR of PAI-1 mRNA was conducted with SYBR Green on a MyiQ real time PCR system, after treatment of the cells with 5 ng/ml TGF-β1 for the indicated times. Results were normalized with the 36B4 expression and were expressed as relative quantity (RQ) (2⋀−ΔΔCt). D, after the stimulation of the NCL cells with TGF-β1 (5 ng/ml) for 90 min, RNA (2 μg) was used for cDNA synthesis with RT2 first standard kit (SuperArray Bioscience, Frederick, MD). The mouse TGF-β/BMP signaling pathway PCR array was performed according to the manufacturer's protocol, using the Profiler PCR array system and the SYBR Green/fluorescein qPCR master mix (SuperArray Bioscience) on a MyiQ real time PCR system (Bio-Rad). Gene expression was compared with the web-based software package for the PCR array system; this software automatically performs all ΔΔCt-based fold-change calculations from the specific uploaded raw threshold cycle data. E, colorimetric diagram with a selection of the 84 TGF-β signaling related genes analyzed. Genes whose transcription is up/down (red/green) 2-fold in gene expression threshold in NCL Cav-1+/+ and NCL Cav-1−/− cells are shown. All results presented are the means ± S.D. of four independent experiments. *, p < 0.01 versus the corresponding unstimulated condition. a, p < 0.01 versus the corresponding value at the same condition in NCL Cav-1+/+.

Article Snippet: The mouse TGF-β/BMP signaling pathway PCR array was performed according to the manufacturer's protocol, using the Profiler PCR array system and the SYBR Green/fluorescein qPCR master mix (SuperArray Bioscience) on a MyiQ real time PCR system (Bio-Rad).

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, SYBR Green Assay, Software, Selection

Journal: The Journal of Biological Chemistry

Article Title: Impairment of Transforming Growth Factor ? Signaling in Caveolin-1-deficient Hepatocytes

doi: 10.1074/jbc.M109.072900

Figure Lengend Snippet: SnoN expression is increased in HCC cells that lack Cav-1. A, mRNA and protein levels of Cav-1 were determined by semiquantitative RT-PCR and Western blot (WB) in THLE-2, CHL, HepG2, and HuH-7 cells. B, HuH-7 cells were stably transfected with Cav-1-GFP expression vector or GFP control vector. The cells that express Cav-1-GFP or GFP were fluorescence-activated cell sorter-subcloned and selected in the presence of G418. Western blot analysis of the protein, Cav-1-GFP, GFP, or Cav-1, expressed after transfection with the pEGFPN1 vector as a control of the process (C+, CHL total extract; C−, HuH-7-WT total extract). C, SnoN expression in two different clones from HuH-7-WT, HuH-7-Cav-1, and HuH7-GFP cells. D, TGF-β-signaling molecules expression analyzed by Western blot in HuH-7-WT and HuH-7-Cav-1 cells after stimulation with TGF-β1 at the indicated times. E, densitometric analysis of SnoN protein levels from D. a.u., arbitrary units. F, HuH-7-WT and HuH-7-Cav-1 cells were transfected with pEGF-N1 and 3TP-lux vectors as described in the legend from Fig. 4A. Results are the means ± S.D. of four independent experiments. *, p < 0.01 versus the corresponding unstimulated. a, p < 0.01 versus the corresponding value at the same condition in HuH-7-Cav-1.

Article Snippet: The mouse TGF-β/BMP signaling pathway PCR array was performed according to the manufacturer's protocol, using the Profiler PCR array system and the SYBR Green/fluorescein qPCR master mix (SuperArray Bioscience) on a MyiQ real time PCR system (Bio-Rad).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Fluorescence, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: Impairment of Transforming Growth Factor ? Signaling in Caveolin-1-deficient Hepatocytes

doi: 10.1074/jbc.M109.072900

Figure Lengend Snippet: TGF-β and HGF signaling cooperate through SnoN expression to increase the proliferation of Cav-1−/− liver cells after PH. A, representative Western blot and the quantification of c-Met protein levels in total liver extracts from Cav-1+/+ and Cav-1−/− mice after PH. B, c-Met protein levels in NCL Cav-1+/+ and NCL Cav-1−/− cells after treatment with 25 ng/ml HGF for 2 h. C, induction of SnoN protein expression by treatment of the NCL cells with HGF for the indicated times. A representative Western blot and a densitometric analysis of the results are shown. D, mRNA PAI-1 levels were measured by qPCR after inhibition of SnoN expression with a specific siRNA or treatment with a scrambled control siRNA. NCL Cav-1+/+ and NCL Cav-1−/− cells were transfected during 6 h with 20 pmol of SnoN siRNA by using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions before TGF-β1 (5 ng/ml) and HGF (25 ng/ml) stimulation for 90 min. E, after transfection of the cells with siRNA and extraction of the mRNA with TRIzol used in D, SnoN protein levels were obtained from organic phase of the TRIzol extraction and analyzed by Western blot. F, 48 h after siRNA transfection, 10 × 103 cells were seeded in a 24-multiwell plate and treated with TGF-β1 (5 ng/ml). The cells were trypsinized, stained with trypan blue, and counted at the indicated times. Results are the means ± S.D. of four independent experiments. *, p < 0.01 versus the corresponding value at 0 time (C) or the corresponding unstimulated data (D). a, p < 0.01 versus the corresponding value at the indicated time (C) or stimulated data in NCL Cav-1+/+(D). b, p < 0.01 versus the corresponding untreated data at 72 h (F). Blots were normalized with p85 for total extracts. a.u., arbitrary units.

Article Snippet: The mouse TGF-β/BMP signaling pathway PCR array was performed according to the manufacturer's protocol, using the Profiler PCR array system and the SYBR Green/fluorescein qPCR master mix (SuperArray Bioscience) on a MyiQ real time PCR system (Bio-Rad).

Techniques: Expressing, Western Blot, Inhibition, Transfection, Staining

Journal: The Journal of Biological Chemistry

Article Title: Impairment of Transforming Growth Factor ? Signaling in Caveolin-1-deficient Hepatocytes

doi: 10.1074/jbc.M109.072900

Figure Lengend Snippet: TGF-β signaling in Cav-1-deficient hepatocytes. In the absence of Cav-1, TβRs delocalize between the membrane and the cytoplasm, leading to a TGF-β-deficient signaling. Lesser activated Smad2/3 translocates to the nucleus, and the higher levels of SnoN impair the transcription of growth-arrest genes and enhance hepatocyte proliferation in the process of liver regeneration. In the presence of Cav-1, TGF-β signaling is sufficient to maintain the balance between cell proliferation and cell growth arrest characteristic of regenerating liver.

Article Snippet: The mouse TGF-β/BMP signaling pathway PCR array was performed according to the manufacturer's protocol, using the Profiler PCR array system and the SYBR Green/fluorescein qPCR master mix (SuperArray Bioscience) on a MyiQ real time PCR system (Bio-Rad).

Techniques: