Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: A-485 alleviates fibrosis and apoptosis in kidney by disrupting tandem activation of acetylation and phosphorylation on STAT3.

doi: 10.1016/j.biopha.2025.118217

Figure Lengend Snippet: Fig. 2. Effects of A-485 on the profibrotic phenotype induced by TGF-β1 in HK-2 cells. (A) The representative western blot images and quantification of FN, Col-I, E- cad and α-SMA in cultured HK-2 cells (n = 3). (B) Micrographs of α-SMA and E-cad immunofluorescence staining in HK-2 cells (n = 6). (C-D) The quantification of α-SMA and E-cad immunofluorescence staining in HK-2 cells. Values are expressed as means ± SEM. *p < 0.05, * *p < 0.01. FN, fibronectin; Col-I, collagen I; E-cad, E-Cadherin; α-SMA, α-smooth muscle actin.

Article Snippet: The antibodies used in the present study are listed as below: antibodies against Bcl-2 (ab196495), Collagen I (ab34710); antibodies against STAT3 (10253–2-AP), Bax (50599–2-Ig), α-SMA (14395–1-AP), Fibronectin (15613–1-AP), E-Cadherin (20874–1-AP) and GAPDH (10494–1-AP) (Proteintech, Chicago, IL, USA); antibodies against Acetyl-STAT3 (Lys685) (#2523), Cleaved Caspase-3 (#9661), p300 (#67621), CBP(#74384SF), Acetylated-Lysine (#9441), PhosphoSTAT3 (S727) (#49081) and Phospho-STAT3 (Y705) (#9145) (Cell Signaling Technology, Beverly, MA, USA); antibodies against NDUFS1 (ER1803–08) and SDHB (ER1803–63) (Hua’an Biotechnology, Hangzhou, China); antibody against COX4 (sc-517553) (Santa Cruz Biotechnology, Santa Cruz, CA); goat anti-rabbit or mouse IgG-HRP secondary antibodies, TRITC-labelled goat anti-rabbit secondary antibody and FITC-labelled goat anti-rabbit or mouse IgG secondary antibodies (Zhongshan Goldenbridge Biotechnology, Beijing, China); Inhibitor A-485 (1889279–16–6) (GlpBio, Montclair, USA).

Techniques: Western Blot, Cell Culture, Immunofluorescence, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: A-485 alleviates fibrosis and apoptosis in kidney by disrupting tandem activation of acetylation and phosphorylation on STAT3.

doi: 10.1016/j.biopha.2025.118217

Figure Lengend Snippet: Fig. 4. Effects of A-485 on UUO-induced profibrotic proteins in mouse kidney. (A-B) The representative western blot images and quantification of FN, Col-I, E-cad and α-SMA in mice (n = 6). (C) Representative micrographs of FN, Col-I and E-cad immunohistochemical staining in mice. (D-F) The quantification of FN, Col-I and E- cad expression detected by immunohistochemical staining (n = 5). Values are expressed as means ± SEM. * *p < 0.01. sham, sham operation; UUO: unilateral ureteral obstruction; FN, fibronectin; Col-I, collagen I; E-cad, E-Cadherin; α-SMA, α-smooth muscle actin.

Article Snippet: The antibodies used in the present study are listed as below: antibodies against Bcl-2 (ab196495), Collagen I (ab34710); antibodies against STAT3 (10253–2-AP), Bax (50599–2-Ig), α-SMA (14395–1-AP), Fibronectin (15613–1-AP), E-Cadherin (20874–1-AP) and GAPDH (10494–1-AP) (Proteintech, Chicago, IL, USA); antibodies against Acetyl-STAT3 (Lys685) (#2523), Cleaved Caspase-3 (#9661), p300 (#67621), CBP(#74384SF), Acetylated-Lysine (#9441), PhosphoSTAT3 (S727) (#49081) and Phospho-STAT3 (Y705) (#9145) (Cell Signaling Technology, Beverly, MA, USA); antibodies against NDUFS1 (ER1803–08) and SDHB (ER1803–63) (Hua’an Biotechnology, Hangzhou, China); antibody against COX4 (sc-517553) (Santa Cruz Biotechnology, Santa Cruz, CA); goat anti-rabbit or mouse IgG-HRP secondary antibodies, TRITC-labelled goat anti-rabbit secondary antibody and FITC-labelled goat anti-rabbit or mouse IgG secondary antibodies (Zhongshan Goldenbridge Biotechnology, Beijing, China); Inhibitor A-485 (1889279–16–6) (GlpBio, Montclair, USA).

Techniques: Western Blot, Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: Fibronectin , REAL555 , 50 , 130-122-864 , PE , Miltenyi Biotec.

Techniques: Imaging

Journal: Scientific Reports

Article Title: TALENs-directed knockout of the full-length transcription factor Nrf1α that represses malignant behaviour of human hepatocellular carcinoma (HepG2) cells

doi: 10.1038/srep23775

Figure Lengend Snippet: All pairs of F/R primers used for real-time qPCR.

Article Snippet: Rabbit polyclonal antibodies against MMP9, SNAI1, p16 and Vimentin were bought from BioSynthesis (Beijing, China), whilst other rabbit antibodies against α-Catenin, CDK2, CDK6, Cyclin D1, SNAI2, FN1, Rb1, p53 and p21 were purchased from BosTer (Wuhan, China).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Activin A and Cell-Surface GRP78 Are Novel Targetable RhoA Activators for Diabetic Kidney Disease

doi: 10.3390/ijms22062839

Figure Lengend Snippet: RhoA mediates the profibrotic effects of activins in diabetic kidney disease (DKD). ( A ) Activin A (20 ng/mL, 3 h) and activin B (20 ng/mL, 3 h) both activated RhoA in mesangial cells (MC) ( n = 2). ( B ) Activation of downstream Rho-kinase was also seen, assessed by phosphorylation of myosin phosphatase target (MYPT) (pMYPT Thr696) in MC ( n = 6–8 * p < 0.05). ( C ) Rho-kinase inhibition with Y27632 (10 μM) or HA1077 (10μM) prevented activin A (20 ng/mL)-induced α-smooth muscle actin (αSMA) promoter luciferase activation ( n = 6–11 *** p < 0.001, **** p < 0.0001) and ( D ) protein expression of both αSMA and the fibrotic protein fibronectin (FN) ( n = 4, * p < 0.05, ** p < 0.01). ( E ) The activin inhibitor follistatin (FST, 500 ng/mL) prevented high glucose (HG) (3 h)-induced RhoA activation ( n = 2) and ( F ) Rho-kinase activation ( n = 3, * p < 0.05, ** p < 0.01). ( G ) RhoA activation in kidneys was also significantly inhibited by follistatin in type 1 diabetic Akita mice, as assessed by IHC with an antibody which specifically detects active RhoA ( n = 4, * p < 0.05, ** p < 0.01).

Article Snippet: The following antibodies were used for immunoblotting: α-smooth muscle actin (αSMA) (Pierce, 1:5000), phosphorylated myosin phosphatase target (pMYPT) Thr696 (Cell Signaling; 1:1000), fibronectin (FN) (BD Biosciences, 1:1000), total RhoA (Santa Cruz, 1:500), MTJ-1 (Santa Cruz, 1:1000), GRP78 (BD Biosciences, 1:1000), PDGFRβ (Cedarlane, 1:1000), vinculin (Sigma, 1:1000) and tubulin (Santa Cruz, 1:5000).

Techniques: Activation Assay, Inhibition, Luciferase, Expressing

Journal: Nature Communications

Article Title: Fibronectin-guided migration of carcinoma collectives

doi: 10.1038/ncomms14105

Figure Lengend Snippet: ( a ) Representative staining of total FN in histospots (600 μm) from HNSCC TMAs scored from 0 to 3. ( b ) Kaplan–Meier estimates of overall and DFS stratified by the dichotomized score of FN expression (low=score 0–1 versus high=score 2–3) in stage I–II (green and blue curves) and stage III–IV (red and black curves) tumours. P -values are from a log-rank test. ( c ) Representative staining of EDA-containing FN in a histospot. Scale bar, 100 μm. ( d ) Western blot analyses of FN expression in lysates of tissue samples from 60 human HNSCC (see Methods). Each blot includes six to eight tumour samples (patient numbers indicated above blots) and a control (Cont) lysate (40 μg, CAL33 cells). Actin staining provides a control of sample integrity and cellularity of tumour samples. ( e ) Western analysis of conditioned medium (top blots) and lysates (bottom blots) from HNSCC lines (CAL33, CAL27, Detroit 562, CAL60 and CAL166), TIF, CAFs and normal human fibroblasts (NF). Equivalent amounts of serum-containing culture medium (10% FCS for tumour cells and 20% FCS for TIFs) were deposited to determine levels of serum-borne FN. Erk1/2 expression in cell lysates was monitored as loading control.

Article Snippet: The following antibodies were used: polyclonal anti-Smad2, anti-phospho-Smad2 (Ser465/467), anti-Smad3 and anti-phospho-Smad3 (Ser423/425) from Cell Signalling Technology (Beverly, MA); monoclonal anti-FN and anti-E-cadherin from BD Biosciences (Le Pont de Claix, France); monoclonal anti-TNC (clone BC24), α-smooth muscle actin (clone 1A4), anti-β actin (clone AC-15) and polyclonal anti-periostin from Sigma-Aldrich; monoclonal anti-collagen I, blocking monoclonal α9β1 antibody (cloneY9A2), rabbit anti-pHis (Ser10) and anti-TGFBI from Abcam (Cambridge, MA); anti-FN-EDA (clone IST-9) from Sirius Biotech (Genoa, Italy); anti-ERK1 (clone C-16) from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-FN, monoclonal anti-αvβ3 integrin (clone LM609), monoclonal anti-α5β1 integrin (MAB1999), blocking monoclonal anti-α5β1 integrin (clone JBS5), monoclonal anti-αvβ5 integrin (clone P1F6), monoclonal anti-αvβ6 integrin (clone E7P6), monoclonal anti-collagen VI, anti-cortactin p80/85 and monoclonal anti-α9β1 integrin were from Millipore (Billerica, MA); anti-β1 integrin (clone lia1/2) from GenWay Biotech (San Diego, CA); blocking monoclonal anti-β1 (clone P5D2) from R&D systems; polyclonal anti-α9 integrin from Thermo Scientific (Rockford, IL); and mouse monoclonal anti-CD31 from DAKO Corp. (Carpinteria, CA).

Techniques: Staining, Expressing, Western Blot

Journal: Nature Communications

Article Title: Fibronectin-guided migration of carcinoma collectives

doi: 10.1038/ncomms14105

Figure Lengend Snippet: ( a ) Scheme of the matrisome analysis workflow. ( b ) Left panels: representative immunofluoresence staining of α-smooth muscle actin (α-SMA) and F-actin in CAFs (top) and TIFs (bottom). Scale bar, 20 μm. Right panels: co-staining of total FN (green) and FN-EDB or FN-EDA/FN (red) in CAFs (top) and TIFs (bottom). Scale bar, 50 μm. Similar stainings were obtained for CAF1 and CAF2 preparations. ( c ) Top 20 ECM glycoproteins identified by mass spectrometry in cell-derived matrices produced by TIFs or by CAFs isolated from two different tumours. See for complete list of matrisome core proteins and matrisome-associated proteins. ( d ) Quantitative PCR analysis of transcripts encoding all FN isoforms (FN1), or isoforms harbouring the EDB and EDA, relative to GAPDH mRNA isolated from confluent TIFs. Results show mean ±s.d. from three independent experiments. ( e ) Comparison of second-harmonic generation (SHG) and immunofluorescence staining of TNC, FN, Col VI and Col I in TIF-derived matrix by two-photon laser microscopy. Representative images are from the same focal plane ( n ≥3 fields per staining from at least three matrix preparations). Scale bar, 50 μm.

Article Snippet: The following antibodies were used: polyclonal anti-Smad2, anti-phospho-Smad2 (Ser465/467), anti-Smad3 and anti-phospho-Smad3 (Ser423/425) from Cell Signalling Technology (Beverly, MA); monoclonal anti-FN and anti-E-cadherin from BD Biosciences (Le Pont de Claix, France); monoclonal anti-TNC (clone BC24), α-smooth muscle actin (clone 1A4), anti-β actin (clone AC-15) and polyclonal anti-periostin from Sigma-Aldrich; monoclonal anti-collagen I, blocking monoclonal α9β1 antibody (cloneY9A2), rabbit anti-pHis (Ser10) and anti-TGFBI from Abcam (Cambridge, MA); anti-FN-EDA (clone IST-9) from Sirius Biotech (Genoa, Italy); anti-ERK1 (clone C-16) from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-FN, monoclonal anti-αvβ3 integrin (clone LM609), monoclonal anti-α5β1 integrin (MAB1999), blocking monoclonal anti-α5β1 integrin (clone JBS5), monoclonal anti-αvβ5 integrin (clone P1F6), monoclonal anti-αvβ6 integrin (clone E7P6), monoclonal anti-collagen VI, anti-cortactin p80/85 and monoclonal anti-α9β1 integrin were from Millipore (Billerica, MA); anti-β1 integrin (clone lia1/2) from GenWay Biotech (San Diego, CA); blocking monoclonal anti-β1 (clone P5D2) from R&D systems; polyclonal anti-α9 integrin from Thermo Scientific (Rockford, IL); and mouse monoclonal anti-CD31 from DAKO Corp. (Carpinteria, CA).

Techniques: Staining, Mass Spectrometry, Derivative Assay, Produced, Isolation, Real-time Polymerase Chain Reaction, Immunofluorescence, Microscopy

Journal: Nature Communications

Article Title: Fibronectin-guided migration of carcinoma collectives

doi: 10.1038/ncomms14105

Figure Lengend Snippet: ( a ) Representative phase contrast images of HNSCC lines plated on plastic or TIF-derived ECM for 24 h. Scale bar, 150 μm. ( b ) Analysis of CAL33 cells on TIF-derived ECM by laser scanning confocal microscopy: (top) three-dimensional rendering of Dapi-stained nuclei and FN-EDA-stained ECM, (bottom) single projections of E-cadherin in intercellular junctions (left) and in tumour cell-derived vesicles sequestered in the ECM (right). Scale bar, 20 μm. ( c ) Migration of non-dividing CAL33 cells ( n ≥100) in cohorts was monitored by time-lapse videomicroscopy, 12 h after seeding on plastic or TIF-derived ECM. Images were acquired every 5 min for 24 h. Representative tracings (denoted by different grey levels) from origin of CAL33 cell cohort migration on plastic (No coat) or ECM are shown. ( d ) Tracks with different directionality ratios were simulated (mean square displacement (MSD) versus time), as described in Methods, and analysed in the same way as experimental data. ( e ) Histograms depict the velocity and directionality of cell movement on plastic (No coat) or ECM. For directionality, the confidence interval is measured as defined by MATLAB in the fit function (see ‘Analysis of cell migration' in Methods). Statistically significant data are indicated by * P <0.05, ** P <0.01, *** P <0.001 or **** P <0.0001. If no statistical difference, error bars are shown at 95%. Results from a representative experiment, of at least three, are shown.

Article Snippet: The following antibodies were used: polyclonal anti-Smad2, anti-phospho-Smad2 (Ser465/467), anti-Smad3 and anti-phospho-Smad3 (Ser423/425) from Cell Signalling Technology (Beverly, MA); monoclonal anti-FN and anti-E-cadherin from BD Biosciences (Le Pont de Claix, France); monoclonal anti-TNC (clone BC24), α-smooth muscle actin (clone 1A4), anti-β actin (clone AC-15) and polyclonal anti-periostin from Sigma-Aldrich; monoclonal anti-collagen I, blocking monoclonal α9β1 antibody (cloneY9A2), rabbit anti-pHis (Ser10) and anti-TGFBI from Abcam (Cambridge, MA); anti-FN-EDA (clone IST-9) from Sirius Biotech (Genoa, Italy); anti-ERK1 (clone C-16) from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-FN, monoclonal anti-αvβ3 integrin (clone LM609), monoclonal anti-α5β1 integrin (MAB1999), blocking monoclonal anti-α5β1 integrin (clone JBS5), monoclonal anti-αvβ5 integrin (clone P1F6), monoclonal anti-αvβ6 integrin (clone E7P6), monoclonal anti-collagen VI, anti-cortactin p80/85 and monoclonal anti-α9β1 integrin were from Millipore (Billerica, MA); anti-β1 integrin (clone lia1/2) from GenWay Biotech (San Diego, CA); blocking monoclonal anti-β1 (clone P5D2) from R&D systems; polyclonal anti-α9 integrin from Thermo Scientific (Rockford, IL); and mouse monoclonal anti-CD31 from DAKO Corp. (Carpinteria, CA).

Techniques: Derivative Assay, Confocal Microscopy, Staining, Migration