Journal: Biomedicines

Article Title: Cell Type-Specific Anti-Adhesion Properties of Peritoneal Cell Treatment with Plasma-Activated Media (PAM)

doi: 10.3390/biomedicines10040927

Figure Lengend Snippet: PAM treatment attenuates protein biosynthesis and the secretion of pro-adhesive factors. Fibroblasts were incubated with 1:2 diluted PAM for 4 h and analyzed after indicated timepoints. ( A ) Representative IF microscopy after staining with anti-5mC antibodies and ( B ) relative genomic methylation level per nucleus (number of foci normalized to the control); the scale bar equals 10 µm. ( C ) DNMT activity level per cell. ( D ) DigiWest protein profiles of pro-adhesive factors collagen I, FGF receptor and FGF-10 and fibronectin relative to controls. ( E ) Cytokine multiplex assay of GM-CSF and IL-1b in fibroblast supernatants relative to control. ( F , G ) Representative IF microscopy of fibronectin ( F ). Scale bars represent 100 and 10 µm, respectively. ( G , H ) Representative Western blot of fibronectin ( G ), and relative of fibronectin expression ( H ) (analyzed from ( G )). ( I , J ) Representative IF microscopy of collagen I ( I ). Scale bars represent 100 and 10 µm, respectively. ( J , K ) Representative Western blot of collagen I ( J ), and relative collagen I expression ( K ) (analyzed from ( J )). ( L ) Relative extracellular hydroxyproline expression. ( M ) Relative MMP-2 expression. ( N ) Relative expression of TGF β. Results are expressed as mean ± SD; * p < 0.05 as determined by paired t -test.

Article Snippet: After washing, membranes were incubated overnight at 4 °C with primary antibodies under constant agitation: fibronectin (1:1000 in 0.1% BSA, abcam), collagen I-alpha antibody (1:1000 in 0.1% BSA, NovusBiologicals, Littleton, CO, USA) and GAPDH (1:1000 in 0.1% BSA, Cell Signaling 14C10).

Techniques: Incubation, Microscopy, Staining, Methylation, Activity Assay, Multiplex Assay, Western Blot, Expressing

Journal: Life

Article Title: The Inhibitory Effect of Noscapine on the In Vitro Cathepsin G-Induced Collagen Expression in Equine Endometrium

doi: 10.3390/life11101107

Figure Lengend Snippet: Levels of significance ( p values) for two- and three-way interactions between estrous cycle phases, treatment time, and cathepsin G (CAT) or noscapine (NOSC) treatments in the analyses of relative transcript COL1A2 gene and COL1 protein relative abundance. The results were considered significant at p < 0.05.

Article Snippet: The COL1 primary antibody (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4 °C and previously validated to equine endometrium by Rebordão et al. [ ].

Techniques: Isolation

Journal: Life

Article Title: The Inhibitory Effect of Noscapine on the In Vitro Cathepsin G-Induced Collagen Expression in Equine Endometrium

doi: 10.3390/life11101107

Figure Lengend Snippet: Inhibition of cathepsin G (0.1 or 1 µg/mL) by noscapine (NOSC; 45 µg/mL) on relative collagen type I alpha 2 chain ( COL1A2 ) mRNA transcription and collagen type I (COL1) protein relative abundance in mare endometrial explants, regardless of estrous cycle phase and time of treatment. Results were considered significant at p < 0.05 and are displayed as least square means ± SEM. Different superscript letters indicate significant differences between CAT concentrations (a, b: CAT 0.1 µg/mL ≠ CAT 1 µg/mL, p < 0.05; c, d: CAT 0.1 µg/mL + NOSC ≠ CAT 1 µg/mL + NOSC, p < 0.05). Asterisks alone represent significant differences relative to the respective control and asterisks above the connecting lines indicate significant differences between treatments (** p < 0.01; *** p < 0.001).

Article Snippet: The COL1 primary antibody (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4 °C and previously validated to equine endometrium by Rebordão et al. [ ].

Techniques: Inhibition, Control

Journal: Life

Article Title: The Inhibitory Effect of Noscapine on the In Vitro Cathepsin G-Induced Collagen Expression in Equine Endometrium

doi: 10.3390/life11101107

Figure Lengend Snippet: Effect of cathepsin G (CAT; 0.1 or 1 µg/mL), noscapine (NOSC; 45 µg/mL), or CAT (0.1 or 1 µg/mL) + NOSC (45 µg/mL) treatments in explants of mare endometrium from follicular phase (FP) or mid-luteal phase (MLP) for 24 or 48 h on relative collagen type I alpha 2 chain ( COL1A2 ) mRNA transcription ( A , B ) and collagen type I (COL1) protein relative abundance ( C , D ). Results were considered significant at p < 0.05 and shown as least square means ± SEM. Asterisks alone represent significant differences relative to the respective control and asterisks above connecting lines indicate significant differences of CAT + NOSC treatment relative to the respective CAT-treated group (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The COL1 primary antibody (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4 °C and previously validated to equine endometrium by Rebordão et al. [ ].

Techniques: Control

Journal: Life

Article Title: The Inhibitory Effect of Noscapine on the In Vitro Cathepsin G-Induced Collagen Expression in Equine Endometrium

doi: 10.3390/life11101107

Figure Lengend Snippet: Effect of cathepsin G (CAT; 0.1 or 1 μg/mL), noscapine (NOSC; 45 μg/mL), or CAT (0.1 or 1 μg/mL) + NOSC (45 μg/mL) treatments on relative collagen type I alpha 2 chain ( COL1A2 ) mRNA transcription ( A ) and collagen type I (COL1) protein relative abundance ( B ) in equine endometrial explants treated for 24 or 48 h, regardless of estrous cycle phase. Results are shown as least square means ± SEM and considered significant at p < 0.05. Asterisks above connecting lines indicate significant differences of the same treatment between time of treatment or differences between different concentrations of CAT at the same time of treatment (* p < 0.05; ** p < 0.01).

Article Snippet: The COL1 primary antibody (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4 °C and previously validated to equine endometrium by Rebordão et al. [ ].

Techniques:

Journal: Life

Article Title: The Inhibitory Effect of Noscapine on the In Vitro Cathepsin G-Induced Collagen Expression in Equine Endometrium

doi: 10.3390/life11101107

Figure Lengend Snippet: Effect of cathepsin G (CAT; 0.1 or 1 μg/mL), noscapine (NOSC; 45 μg/mL), or CAT (0.1 or 1 μg/mL) + NOSC (45 μg/mL) treatments on relative collagen type I alpha 2 chain ( COL1A2 ) mRNA transcription ( A ) and collagen type I (COL1) protein relative abundance ( B ) in explants of mare endometrium from follicular (FP) or mid-luteal (MLP) phases, regardless of treatment time. Results are shown at least square means ± SEM and considered significant at p < 0.05. Asterisks above connecting lines indicate significant differences of the same treatment between estrous cycle phase or differences between different concentrations of CAT at the same estrous cycle phase (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The COL1 primary antibody (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4 °C and previously validated to equine endometrium by Rebordão et al. [ ].

Techniques:

Journal: International Wound Journal

Article Title: Machine learning and single‐cell transcriptome profiling reveal regulation of fibroblast activation through THBS2/TGFβ1/P‐Smad 2/3 signalling pathway in hypertrophic scar

doi: 10.1111/iwj.14481

Figure Lengend Snippet: THBS2 promotes the proliferation, migration and expression of fibrotic marker protein. (A) qRT‐PCR showed that CTSK, CTHRC1 and THBS2 were expressed in HS. (B) qRT‐PCR showed that THBS2 was knocked down by siRNA in scar fibroblasts. (C) qRT‐PCR showed that the expression of Col1, Col3 and αSMA in fibroblasts with low THBS2 knockdown was decreased. (D) Western blotting showed that the expression of Col1, Col3 and αSMA decreased in fibroblasts with decreased THBS2 expression. (E) Cellular immunofluorescence showed decreased αSMA expression in fibroblasts with THBS2 knockdown. (F) Scratch test showed that the migration rate of fibroblasts with low THBS2 was decreased (scale bar = 100 μm). (G) CCK8 assay showed that the proliferation of THBS2 knockdown fibroblasts decreased. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. HS, hypertrophic scar; NS, normal skin.

Article Snippet: Subsequently, the PVDF membranes were washed with TBST before being incubated overnight at 4°C with the following rabbit anti‐human primary antibodies: GAPDH (10494‐1‐AP, Sanying, Wuhan, China, 1:1000), THBS2 (ab84469, Abcam, 1:1000), Col1 (14695‐1‐AP, Sanying, Wuhan, China, 1:1000), Col3 (22734‐1‐AP, Sanying, Wuhan, China, 1:1000), αSMA (14395‐1‐AP, Sanying, Wuhan, China, 1:1000), TGFβ1 (ab215715, Abcam), P‐Smad2/3 (8828 s, Cell Signalling Technology, 1:1000), Smad2/3 (8685s, Cell Signalling Technology, 1:1000).

Techniques: Migration, Expressing, Marker, Quantitative RT-PCR, Knockdown, Western Blot, Immunofluorescence, CCK-8 Assay

Journal: International Wound Journal

Article Title: Machine learning and single‐cell transcriptome profiling reveal regulation of fibroblast activation through THBS2/TGFβ1/P‐Smad 2/3 signalling pathway in hypertrophic scar

doi: 10.1111/iwj.14481

Figure Lengend Snippet: THBS2 activates scar fibroblasts through the TGFβ1/P‐Smad2/3 pathway. (A) GSEA plot demonstrates the association between THBS2 and the TGF‐β pathway. (B) qRT‐PCR showed the mRNA expression levels of Col1, Col3, αSMA and TGFβ1 in the control group, THBS2 (200 ng/mL), THBS2 (200 ng/mL) + SB‐431542 (10 ng/mL), THBS2 (200 ng/mL) + SB‐431542 (10 ng/mL) + TGFβ1 (10 ng/mL) treated fibroblasts. (C) Protein expression levels of Col1, Col3, αSMA and P‐Smad2/3 in the control group, THBS2, THBS2 + SB‐431542, THBS2 + SB‐431542 + TGFβ1‐treated fibroblasts by western blotting. (D) The proliferation ability of four groups of scar fibroblasts was tested by CCK8 proliferation test. (^: comparison between control and THBS2 groups; *: comparison between THBS2 and THBS2 + SB‐431542 groups). (E) The migration ability of four groups of scar fibroblasts was tested by cell scratch test (scale bar = 100 μm). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Subsequently, the PVDF membranes were washed with TBST before being incubated overnight at 4°C with the following rabbit anti‐human primary antibodies: GAPDH (10494‐1‐AP, Sanying, Wuhan, China, 1:1000), THBS2 (ab84469, Abcam, 1:1000), Col1 (14695‐1‐AP, Sanying, Wuhan, China, 1:1000), Col3 (22734‐1‐AP, Sanying, Wuhan, China, 1:1000), αSMA (14395‐1‐AP, Sanying, Wuhan, China, 1:1000), TGFβ1 (ab215715, Abcam), P‐Smad2/3 (8828 s, Cell Signalling Technology, 1:1000), Smad2/3 (8685s, Cell Signalling Technology, 1:1000).

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, Comparison, Migration