Journal: bioRxiv

Article Title: Defining the role of fibroblasts in skin expansion

doi: 10.1101/2025.03.18.643853

Figure Lengend Snippet: a-c – Expression of Col1a1, Col3a1 and Fn1 in fibroblasts from scRNAseq, per mouse, at D1D4. d – Heatmap showing all differentially expressed genes (DEG) from bulk RNAseq analysis of sorted fibroblasts. Relative gene expression (Rel. gene exp.) is shown. e,f – Venn diagrams showing numbers of upregulated and downregulated genes respectively from bulk RNAseq of sorted Pdgfrα+ and Cd34+ fibroblasts in EXP compared to CTRL at D1-4. g – Heatmaps of DEG from bulk RNAseq analysis of sorted Pdgfrα+ and Cd34+ fibroblasts. Relative gene expression (Rel. gene exp.) is shown for the indicated gene ontology (GO) term, with red indicating high expression, and blue indicating low expression. h-j – Relative expression of the indicated genes in sorted fibroblasts by qPCR at D2. Unpaired one-tailed Mann-Whitney U-tests, n=3 mice per condition. Data are presented as mean +/- SD. k – Heatmap of DEG within the gene ontology (GO) term for Ribosome biogenesis. Relative gene expression is shown, with red indicating high expression, and blue indicating low expression. l – Heatmap of DEG within the Reactome term for signalling by Wnt. Relative gene expression is shown, with red indicating high expression, and blue indicating low expression. m – Heatmap of DEG within the Reactome term for YAP1/TAZ-stimulated gene expression. Relative gene expression is shown, with red indicating high expression, and blue indicating low expression. n – Immunofluorescence for Yap (White), Vim (green) and Hoechst (blue) in D4 CTRL and EXP skin. Pink dotted lines separate the dermis from the epidermis and hair follicles. Yellow arrows indicate nuclear Yap nuclear localization. Scale bars=50 μm. o – Quantification of nuclear Yap in Vim+ dermal cells from m . Unpaired two-tailed MannWhitney U-test, n=3 mice per condition, means of 3 measurements per mouse are plotted as dots. Data are presented as mean +/- SD. p – Relative expression of Ccn2 ( Ctgf ) in sorted fibroblasts by qPCR at D2. Unpaired one-tailed Mann-Whitney U-tests, n=3 mice per condition. Data represent mean +/- SD.

Article Snippet: The following primary antibodies were used: anti-Col4A1 (600-101-MN4, Rockland, 1:400), anti-Fn1 (66042-1-Ig, Proteintech, 1:1000).

Techniques: Expressing, Gene Expression, One-tailed Test, MANN-WHITNEY, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Defining the role of fibroblasts in skin expansion

doi: 10.1101/2025.03.18.643853

Figure Lengend Snippet: a,b – Serial cultivation of normal human keratinocytes on SQ-FL (orange line), as compared to no feeder layer conditions, with fresh KGM medium (FM-noFL, grey line) or fibroblast conditioned medium (CM-noFL, green line). Percentage of clonogenic cells was calculated as the ratio between grown colonies and plated cells ( a ). Percentage of aborted colonies was calculated as the ratio between the colonies scored as aborted and the number of clonogenic cells ( b ). n=2 independent biological replicates. Data are presented as mean +/− SEM. c – Total amount of cells obtained at subconfluence during serial cultivation of normal human keratinocytes (NHK) on SQ-FL (orange line) as compared to FM-noFL (grey line) or CM-noFL, (blue line). n=2 independent biological replicates. Data are presented as mean +/− SEM. d – Western analysis of total cell extracts from cultures generated by NHKs on SQ-FL, FM-noFL or CM-noFL. One of two representative experiments is shown. e – Numbers of differentially expressed proteins (DEP) from mass spectrometry analysis of two independent experiments (1°EXP and 2°EXP). Upregulated (DEP-UP), downregulated (DEP-DOWN) and total (TOT DEP) protein numbers are plotted. f – Bar showing log2 Fold change of some of the downregulated or upregulated proteins of interest. g , h – Graphs showing downregulated ( g ) and upregulated ( h ) cellular components in mass spectrometry analysis of SQ as compared to LQ feeder layers (2°EXP). p values are calculated with one-sided Fisher’s Exact test and corrected for multiple tests with the Benjamini– Hochberg method. Data presented as –log10 of corrected p value (dark blue and dark red bars respectively) and corresponding fold change (light blue and light red bars respectively). i , j – Graphs showing downregulated ( i ) and upregulated ( j ) cellular components in mass spectrometry analysis of SQ as compared to LQ feeder layers (1°EXP). p values are calculated with one-sided Fisher’s Exact test and corrected for multiple tests with the Benjamini– Hochberg method. Data presented as –log10 of corrected p value (dark blue and dark red bars respectively) and corresponding fold change (light blue and light red bars respectively). k – Representative immunofluorescence of the two type of feeder layer stained for COL4A1 (green) and FN1 (red) and nuceli (blue). Scale bar = 50 μm

Article Snippet: The following primary antibodies were used: anti-Col4A1 (600-101-MN4, Rockland, 1:400), anti-Fn1 (66042-1-Ig, Proteintech, 1:1000).

Techniques: Western Blot, Generated, Mass Spectrometry, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Defining the role of fibroblasts in skin expansion

doi: 10.1101/2025.03.18.643853

Figure Lengend Snippet: a – Volcano plot representation of the proteins identified by mass spectrometry analysis of standard quality feeder layers (SQ-FL) compared to low quality feeder layers (LQ-FL). Significantly downregulated proteins are shown in blue, significantly upregulated proteins are shown in red. x-axis shows fold-change (log2(ratio)), y-axis shows proteins’ significance (as Log10 of the corresponding q-value). b – Graph showing downregulated cellular component terms in SQ-FL as compared to LQ-FL. p values are calculated with one-sided Fisher’s Exact test and corrected for multiple tests with the Benjamini–Hochberg method. Data presented as –log10 of the corrected p value (dark blue bars) and corresponding fold changes (light blue bars). c – Graph showing upregulated cellular component terms in SQ-FL as compared to LQ-FL. Pvalues are calculated with one-sided Fisher’s Exact test and corrected for multiple tests with the Benjamini–Hochberg method. Data presented as –log10 of the corrected p value (red bars) and corresponding fold changes (dark red bars). d – Western analysis of total cell extracts from SQ-FL and LQ-FL. One out of three representative experiments is shown. e, f – Quantification of staining intensity related to for Col4a1 ( e ) and Fn1 ( f ). . P value calculated by unpaired t -students test. Data are presented as mean +/- SD. g , h – Serial cultivation of normal human keratinocytes on LQ-FL (blue line) and SQ-FL (orange line); Percentage of clonogenic cells was calculated as the ratio between grown colonies and plated cells ( g ). Percentage of aborted colonies was calculated as the ratio between the colonies scored as aborted and the number of clonogenic cells ( h ). Two-tailed paired Student t-test, n=3 independent biological replicates. Data are presented as mean +/− SEM. i – Total amount of cells obtained at subconfluence during serial cultivation of normal human keratinocytes on LQ-FL (blue line) and SQ-FL (orange line). Two-tailed paired Student t-test, n=3 independent biological replicates. Data are presented as mean +/− SEM. j – Representative images related to g - i showing the indicator dishes derived from NHK cultures, coltured on SQ-FL or LQ-FL at the indicated passage. k – qRT-PCR quantification of the mRNA levels of clonogenic markers ( FOXM1, H1B, ANLN, AURKB ) and differentiation markers ( IVL, SPINK5, TGM1 ) on three human primary keratinocytes cultures grown on SQ-FL (orange bars) and LQ-FL (blue bars). Two-tailed paired Student t-tests, FOXM1 *p=0.040, H1B *p=0.033, ANLN p=0.094, AURKB *p=0.031, IVL p=0.327, SPINK5 p=0.327, TGM1 p=0.327. n=3 independent biological replicates. Data are presented as mean +/− SD. l – Western analysis of total cell extracts from cultures generated by human primary keratinocytes grown on SQ-FL and LQ-FL. One out of three representative experiments is shown.

Article Snippet: The following primary antibodies were used: anti-Col4A1 (600-101-MN4, Rockland, 1:400), anti-Fn1 (66042-1-Ig, Proteintech, 1:1000).

Techniques: Mass Spectrometry, Western Blot, Staining, Two Tailed Test, Derivative Assay, Quantitative RT-PCR, Generated

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibitory Effects of Oxymatrine on Transdifferentiation of Neonatal Rat Cardiac Fibroblasts to Myofibroblasts Induced by Aldosterone via Keap1/Nrf2 Signaling Pathways In Vitro

doi: 10.12659/MSM.915542

Figure Lengend Snippet: Effects of OMT and curcumin on Collagen I, Collagen III, FN, α-SMA, CTGF, and MR induced by ALD in CFBs. CFBs were pretreated with 10 μmol/L curcumin, 1 μmol/L spironolactone (Spiro), 18.9 μmol/L OMT, or 37.8 μmol/L OMT for 2 h, and then exposed to 0.1 μmol/L ALD for 24 h. ( A ) The expression of hydroxyproline content in CFBs supernatant was determined using a commercial kit. Western blot was used to assess expression of ( B, C ) Collagen I, ( D ) Collagen III, ( E ) FN, ( F ) α-SMA, ( G ) CTGF, and ( H ) MR protein in CFBs. ( I ) Immunofluorescent staining for α-SMA (green) and nuclear marker DAPI (blue) in CFBs after indicated treatment (magnification, 200×). Results are presented as the mean ±SEM (* p<0.05 and ** p<0.01 vs. control; # p<0.05 and ## p<0.01 vs. ALD).

Article Snippet: Equivalent amounts of protein samples (20–30 μg) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on an 8% gel and subsequently transferred to polyvinylidene difluoride (PVDF) membranes at 4°C, then blocked with 5% non-fat milk in TBST at room temperature for 2 h. The membranes were incubated with the following primary antibody at 4°C overnight: α-SMA (14395-1-AP, Proteintech, 1: 1000), Fibronectin (66042-1-lg, Proteintech, 1: 5000), CTGF (MD5912-50, Medical Discovery Leader, 1: 1000), Collagen III (MD4405-50, Medical Discovery Leader, 1: 1000), HO-1(66743-1-lg, Proteintech, 1: 1000), and Nrf2 (16396-1-AP, Proteintech, 1: 1000), Keap1(10503-2-AP, Proteintech, 1: 1000), collagen I (14695-1-AP, Proteintech, 1: 1000), MR (21854-1-AP, Proteintech, 1: 1000), LaminB1 (12987-1-AP, Proteintech, 1: 1000), Tubulin (10094-1-AP, Proteintech, 1: 10,000) and GAPDH (60004-1-lg, Proteintech, 1: 10000).

Techniques: Expressing, Western Blot, Staining, Marker, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibitory Effects of Oxymatrine on Transdifferentiation of Neonatal Rat Cardiac Fibroblasts to Myofibroblasts Induced by Aldosterone via Keap1/Nrf2 Signaling Pathways In Vitro

doi: 10.12659/MSM.915542

Figure Lengend Snippet: OMT inhibited ALD-induced cardiac fibrosis depending on Nrf2 signal pathway. ( A, B ) Efficiency test of Nrf2 siRNA. Cardiac fibroblasts were transfected with negative control siRNA (NC) or Nrf2 siRNA, and the total protein of Nrf2 was determined by Western blot. Western blot analysis of ( C, D ) α-SMA, ( E ) CTGF ( F ) Collagen I, and ( G ) Collagen III protein expression in CFBs. ( H ) Immunofluorescent staining for α-SMA (green) and nuclear marker DAPI (blue) in CFBs. Results are presented as the mean ±SEM (* p<0.05 and ** p<0.01 vs. control; # p<0.05 and ## p<0.01 vs. ALD; & p<0.05 vs. OMT+ALD).

Article Snippet: Equivalent amounts of protein samples (20–30 μg) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on an 8% gel and subsequently transferred to polyvinylidene difluoride (PVDF) membranes at 4°C, then blocked with 5% non-fat milk in TBST at room temperature for 2 h. The membranes were incubated with the following primary antibody at 4°C overnight: α-SMA (14395-1-AP, Proteintech, 1: 1000), Fibronectin (66042-1-lg, Proteintech, 1: 5000), CTGF (MD5912-50, Medical Discovery Leader, 1: 1000), Collagen III (MD4405-50, Medical Discovery Leader, 1: 1000), HO-1(66743-1-lg, Proteintech, 1: 1000), and Nrf2 (16396-1-AP, Proteintech, 1: 1000), Keap1(10503-2-AP, Proteintech, 1: 1000), collagen I (14695-1-AP, Proteintech, 1: 1000), MR (21854-1-AP, Proteintech, 1: 1000), LaminB1 (12987-1-AP, Proteintech, 1: 1000), Tubulin (10094-1-AP, Proteintech, 1: 10,000) and GAPDH (60004-1-lg, Proteintech, 1: 10000).

Techniques: Transfection, Negative Control, Western Blot, Expressing, Staining, Marker, Control

Journal: bioRxiv

Article Title: Defining the role of fibroblasts in skin expansion

doi: 10.1101/2025.03.18.643853

Figure Lengend Snippet: a – Volcano plot representation of the proteins identified by mass spectrometry analysis of standard quality feeder layers (SQ-FL) compared to low quality feeder layers (LQ-FL). Significantly downregulated proteins are shown in blue, significantly upregulated proteins are shown in red. x-axis shows fold-change (log2(ratio)), y-axis shows proteins’ significance (as Log10 of the corresponding q-value). b – Graph showing downregulated cellular component terms in SQ-FL as compared to LQ-FL. p values are calculated with one-sided Fisher’s Exact test and corrected for multiple tests with the Benjamini–Hochberg method. Data presented as –log10 of the corrected p value (dark blue bars) and corresponding fold changes (light blue bars). c – Graph showing upregulated cellular component terms in SQ-FL as compared to LQ-FL. Pvalues are calculated with one-sided Fisher’s Exact test and corrected for multiple tests with the Benjamini–Hochberg method. Data presented as –log10 of the corrected p value (red bars) and corresponding fold changes (dark red bars). d – Western analysis of total cell extracts from SQ-FL and LQ-FL. One out of three representative experiments is shown. e, f – Quantification of staining intensity related to for Col4a1 ( e ) and Fn1 ( f ). . P value calculated by unpaired t -students test. Data are presented as mean +/- SD. g , h – Serial cultivation of normal human keratinocytes on LQ-FL (blue line) and SQ-FL (orange line); Percentage of clonogenic cells was calculated as the ratio between grown colonies and plated cells ( g ). Percentage of aborted colonies was calculated as the ratio between the colonies scored as aborted and the number of clonogenic cells ( h ). Two-tailed paired Student t-test, n=3 independent biological replicates. Data are presented as mean +/− SEM. i – Total amount of cells obtained at subconfluence during serial cultivation of normal human keratinocytes on LQ-FL (blue line) and SQ-FL (orange line). Two-tailed paired Student t-test, n=3 independent biological replicates. Data are presented as mean +/− SEM. j – Representative images related to g - i showing the indicator dishes derived from NHK cultures, coltured on SQ-FL or LQ-FL at the indicated passage. k – qRT-PCR quantification of the mRNA levels of clonogenic markers ( FOXM1, H1B, ANLN, AURKB ) and differentiation markers ( IVL, SPINK5, TGM1 ) on three human primary keratinocytes cultures grown on SQ-FL (orange bars) and LQ-FL (blue bars). Two-tailed paired Student t-tests, FOXM1 *p=0.040, H1B *p=0.033, ANLN p=0.094, AURKB *p=0.031, IVL p=0.327, SPINK5 p=0.327, TGM1 p=0.327. n=3 independent biological replicates. Data are presented as mean +/− SD. l – Western analysis of total cell extracts from cultures generated by human primary keratinocytes grown on SQ-FL and LQ-FL. One out of three representative experiments is shown.

Article Snippet: The following primary antibodies were used: anti-IVL 1:5000 from Invitrogen (Ma1-25752, mouse), anti-FOXM1 1:1000 from Cell Signalling Technologies (D3f2b, rabbit), anti-P63α 1:1000 from Cell Signalling Technologies (4892s, rabbit), anti-hGAPDH 1:500 from Sigma-Aldrich (Zrb374, rabbit), antiCOL4 1:1000 from Rockland (600-101-MN4, Goat), anti-FN1 from Proteintech 1:10000 (66042-1-Ig, Mouse), anti-GAPDH 1:500 from Abcam (ab8245, mouse).

Techniques: Mass Spectrometry, Western Blot, Staining, Two Tailed Test, Derivative Assay, Quantitative RT-PCR, Generated