primary human dermal fibroblasts hdf  (ATCC)


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    ATCC primary human dermal fibroblasts hdf
    ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal <t>fibroblasts</t> <t>(HDF)</t> were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.
    Primary Human Dermal Fibroblasts Hdf, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Chloride Homeostasis Regulates cGAS-STING Signaling"

    Article Title: Chloride Homeostasis Regulates cGAS-STING Signaling

    Journal: bioRxiv

    doi: 10.1101/2024.04.08.588475

    ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.
    Figure Legend Snippet: ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.

    Techniques Used: Activation Assay, Cell Culture, Activity Assay, Transfection, Comparison, Expressing, Quantitative RT-PCR, Western Blot, Staining

    normal human dermal nhd fibroblast cell lines  (ATCC)


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    ATCC normal human dermal nhd fibroblast cell lines
    (A) Schematic graph illustrating the timeline for the culture and TGF-β1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and Normal Human Dermal <t>(NHD)</t> <t>fibroblast</t> cells. (B) RUNX1 expression rate in samples treated with TGF-β1 (in red) vs. control for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the two SSc-isolated fibroblast lines at 12 hours after exposure vs. the baseline. (D) The pathway analysis of Reactome gene sets shows the biological pathways and processes that are significantly represented within top DEG genes of SSc-isolated fibroblast lines 12 hours after TGF-β1 treatment vs. the baseline. (E) Fold change expression of TGF-β1 and CBFB in TGF-β1-induced SSc fibroblasts treated with Ro5-3335 ( RUNX1 inhibitor), compared to control. (F) Proliferation curve of NHD fibroblasts in the presence and absence of Ro5-3335. (G–H) The 3D collagen contraction assays, fixed (G) and floating ( H ) models, of NHD fibroblasts treated with Ro5-3335. SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. (Student’s t-test P- value: **0.001–0.01, ****<0.0001 in GraphPad Prism v9)
    Normal Human Dermal Nhd Fibroblast Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "RUNX1 is Expressed in a Subpopulation of Dermal Fibroblasts and Higher RUNX1 Levels are Associated with the Severity of Systemic Sclerosis"

    Article Title: RUNX1 is Expressed in a Subpopulation of Dermal Fibroblasts and Higher RUNX1 Levels are Associated with the Severity of Systemic Sclerosis

    Journal: bioRxiv

    doi: 10.1101/2024.04.03.587966

    (A) Schematic graph illustrating the timeline for the culture and TGF-β1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and Normal Human Dermal (NHD) fibroblast cells. (B) RUNX1 expression rate in samples treated with TGF-β1 (in red) vs. control for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the two SSc-isolated fibroblast lines at 12 hours after exposure vs. the baseline. (D) The pathway analysis of Reactome gene sets shows the biological pathways and processes that are significantly represented within top DEG genes of SSc-isolated fibroblast lines 12 hours after TGF-β1 treatment vs. the baseline. (E) Fold change expression of TGF-β1 and CBFB in TGF-β1-induced SSc fibroblasts treated with Ro5-3335 ( RUNX1 inhibitor), compared to control. (F) Proliferation curve of NHD fibroblasts in the presence and absence of Ro5-3335. (G–H) The 3D collagen contraction assays, fixed (G) and floating ( H ) models, of NHD fibroblasts treated with Ro5-3335. SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. (Student’s t-test P- value: **0.001–0.01, ****<0.0001 in GraphPad Prism v9)
    Figure Legend Snippet: (A) Schematic graph illustrating the timeline for the culture and TGF-β1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and Normal Human Dermal (NHD) fibroblast cells. (B) RUNX1 expression rate in samples treated with TGF-β1 (in red) vs. control for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the two SSc-isolated fibroblast lines at 12 hours after exposure vs. the baseline. (D) The pathway analysis of Reactome gene sets shows the biological pathways and processes that are significantly represented within top DEG genes of SSc-isolated fibroblast lines 12 hours after TGF-β1 treatment vs. the baseline. (E) Fold change expression of TGF-β1 and CBFB in TGF-β1-induced SSc fibroblasts treated with Ro5-3335 ( RUNX1 inhibitor), compared to control. (F) Proliferation curve of NHD fibroblasts in the presence and absence of Ro5-3335. (G–H) The 3D collagen contraction assays, fixed (G) and floating ( H ) models, of NHD fibroblasts treated with Ro5-3335. SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. (Student’s t-test P- value: **0.001–0.01, ****<0.0001 in GraphPad Prism v9)

    Techniques Used: Isolation, Expressing, Positive Control

    normal primary human dermal fibroblasts  (ATCC)


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    ATCC normal primary human dermal fibroblasts
    αSMA gene expression and protein levels are reduced in TGFβ1-activated <t>fibroblasts</t> following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.
    Normal Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Ketotifen directly modifies the fibrotic response of human skin fibroblasts"

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-57776-7

    αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.
    Figure Legend Snippet: αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

    Ketotifen treatment reduced αSMA fibres in TGFβ1-activated fibroblasts. HDFa cells were cultured on poly- d -lysine-coated coverslips and treated for 48 h with media, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A ) Representative images of immunofluorescence staining (200X total magnification) probing for αSMA and DAPI are shown on the different groups of HDFa cells. ( B ) Quantification of αSMA+ staining (green) was plotted per DAPI+ cell (blue). Measurements for each biological replicate were averaged from five microscopic fields of view. Data shown as mean ± SEM. n = 3 per treatment condition. ** p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA, alpha-smooth muscle actin, DAPI, 4′, 6-diamidino-2-phenylindole, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta 1.
    Figure Legend Snippet: Ketotifen treatment reduced αSMA fibres in TGFβ1-activated fibroblasts. HDFa cells were cultured on poly- d -lysine-coated coverslips and treated for 48 h with media, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A ) Representative images of immunofluorescence staining (200X total magnification) probing for αSMA and DAPI are shown on the different groups of HDFa cells. ( B ) Quantification of αSMA+ staining (green) was plotted per DAPI+ cell (blue). Measurements for each biological replicate were averaged from five microscopic fields of view. Data shown as mean ± SEM. n = 3 per treatment condition. ** p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA, alpha-smooth muscle actin, DAPI, 4′, 6-diamidino-2-phenylindole, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta 1.

    Techniques Used: Cell Culture, Immunofluorescence, Staining

    Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.
    Figure Legend Snippet: Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.

    Techniques Used: Expressing

    Ketotifen treatment increased phosphorylation of YAP, decreased TAZ protein levels, and inhibited AKT phosphorylation. HDFa cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A , B ) Gene expression of YAP ( YAP1 ) and TAZ ( WWTR1 ) were assessed using RT-qPCR and normalized to housekeeping gene HPRT . ( C ) Phosphorylated YAP at serine residue 127 and total YAP protein were measured using western blot and semi-quantitative assessments made by normalization to GAPDH. ( D ) TAZ protein was measured and plotted in the same manner. ( E ) Representative images of the blots are shown. ( F ) Phosphorylated AKT at serine residue 473 was measured in TGFβ1-treated fibroblasts, in the presence of ketotifen or AKT inhibitor, MK2206 (left). Representative blot images of phosphorylated AKT are on the right. Full-length blots are available in Supplementary Fig. . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001. αSMA alpha-smooth muscle actin, AKT protein kinase B, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.
    Figure Legend Snippet: Ketotifen treatment increased phosphorylation of YAP, decreased TAZ protein levels, and inhibited AKT phosphorylation. HDFa cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A , B ) Gene expression of YAP ( YAP1 ) and TAZ ( WWTR1 ) were assessed using RT-qPCR and normalized to housekeeping gene HPRT . ( C ) Phosphorylated YAP at serine residue 127 and total YAP protein were measured using western blot and semi-quantitative assessments made by normalization to GAPDH. ( D ) TAZ protein was measured and plotted in the same manner. ( E ) Representative images of the blots are shown. ( F ) Phosphorylated AKT at serine residue 473 was measured in TGFβ1-treated fibroblasts, in the presence of ketotifen or AKT inhibitor, MK2206 (left). Representative blot images of phosphorylated AKT are on the right. Full-length blots are available in Supplementary Fig. . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001. αSMA alpha-smooth muscle actin, AKT protein kinase B, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Techniques Used: Expressing, Quantitative RT-PCR, Residue, Western Blot, Binding Assay

    Ketotifen fumarate treatment impaired TGFβ1-stimulated fibroblast contraction in an in vitro model of fibroblast-populated gel contraction. ( A ) Representative images of fibroblast-populated collagen gels in DF10 (DMEM, with 10% FBS) culture media with or without ketotifen fumarate at 0- and 4 h post-treatment. ( B ) Contracture of activated fibroblast-populated collagen gels was determined and plotted over the course of 24 h as mean gel area in normal or ketotifen-supplemented media. ( C ) TGFβ1 or TGFβ1 and ketotifen fumarate-treated fibroblasts were seeded into collagen gels and contracture measured in normal culture media. ( D ) ACTA2 is significantly reduced in ketotifen-treated TGFβ1-activated fibroblasts compared to TGFβ1 treatment at 4 h. Gene expression data is normalized to housekeeping gene HPRT . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; *** p < 0.001; **** p < 0.0001. DF10 10% FBS-DMEM, DF10-K 10% FBS-DMEM with ketotifen fumarate, TGFβ1 transforming growth factor-beta 1, TK25 transforming growth factor-beta 1 with 25 μM ketotifen.
    Figure Legend Snippet: Ketotifen fumarate treatment impaired TGFβ1-stimulated fibroblast contraction in an in vitro model of fibroblast-populated gel contraction. ( A ) Representative images of fibroblast-populated collagen gels in DF10 (DMEM, with 10% FBS) culture media with or without ketotifen fumarate at 0- and 4 h post-treatment. ( B ) Contracture of activated fibroblast-populated collagen gels was determined and plotted over the course of 24 h as mean gel area in normal or ketotifen-supplemented media. ( C ) TGFβ1 or TGFβ1 and ketotifen fumarate-treated fibroblasts were seeded into collagen gels and contracture measured in normal culture media. ( D ) ACTA2 is significantly reduced in ketotifen-treated TGFβ1-activated fibroblasts compared to TGFβ1 treatment at 4 h. Gene expression data is normalized to housekeeping gene HPRT . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; *** p < 0.001; **** p < 0.0001. DF10 10% FBS-DMEM, DF10-K 10% FBS-DMEM with ketotifen fumarate, TGFβ1 transforming growth factor-beta 1, TK25 transforming growth factor-beta 1 with 25 μM ketotifen.

    Techniques Used: In Vitro, Expressing

    Schematic of the effects of ketotifen fumarate on fibroblast phenotype and associated cellular mechanisms. ( A ) Ketotifen fumarate inhibits the effects of TGFβ1 on fibroblasts, decreasing αSMA gene and protein levels, cytoskeletal-associated genes, and contractility. ( B ) Ketotifen fumarate treatment reduces ACTA2 , CNN1 , and TAGLN , and decreases TAZ gene and protein levels. Increased phosphorylation of YAP at serine residue 127 is also seen, promoting cytosolic localization. Ketotifen also affects the PI3K-AKT pathway by reducing phosphorylation of AKT at serine residue 473. AKT protein kinase B, αSMA alpha-smooth muscle actin, CNN1 calponin 1, PI3K phosphatidylinositol-3-kinase, TAGLN transgelin, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.
    Figure Legend Snippet: Schematic of the effects of ketotifen fumarate on fibroblast phenotype and associated cellular mechanisms. ( A ) Ketotifen fumarate inhibits the effects of TGFβ1 on fibroblasts, decreasing αSMA gene and protein levels, cytoskeletal-associated genes, and contractility. ( B ) Ketotifen fumarate treatment reduces ACTA2 , CNN1 , and TAGLN , and decreases TAZ gene and protein levels. Increased phosphorylation of YAP at serine residue 127 is also seen, promoting cytosolic localization. Ketotifen also affects the PI3K-AKT pathway by reducing phosphorylation of AKT at serine residue 473. AKT protein kinase B, αSMA alpha-smooth muscle actin, CNN1 calponin 1, PI3K phosphatidylinositol-3-kinase, TAGLN transgelin, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Techniques Used: Residue, Binding Assay

    normal human ws1 dermal fibroblasts  (ATCC)


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    ATCC normal human ws1 dermal fibroblasts
    αSMA gene expression and protein levels are reduced in TGFβ1-activated <t>fibroblasts</t> following ketotifen treatment. ( A , B ) HDFa and ( C , D ) <t>WS1</t> fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.
    Normal Human Ws1 Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    normal human ws1 dermal fibroblasts - by Bioz Stars, 2024-04
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    1) Product Images from "Ketotifen directly modifies the fibrotic response of human skin fibroblasts"

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-57776-7

    αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.
    Figure Legend Snippet: αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

    Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.
    Figure Legend Snippet: Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.

    Techniques Used: Expressing

    primary human dermal fibroblasts  (ATCC)


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    ATCC primary human dermal fibroblasts
    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal <t>fibroblasts.</t> At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
    Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors"

    Article Title: Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-54941-w

    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
    Figure Legend Snippet: HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for VEGF, PGF, or ANGPT1, expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
    Figure Legend Snippet: HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for VEGF, PGF, or ANGPT1, expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for FLT-1, or EDN1 expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
    Figure Legend Snippet: HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for FLT-1, or EDN1 expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    primary human dermal fibroblasts phdf  (ATCC)


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    ATCC primary human dermal fibroblasts phdf
    Primary Human Dermal Fibroblasts Phdf, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human dermal fibroblasts hdfa  (ATCC)


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    ATCC human dermal fibroblasts hdfa
    Human Dermal Fibroblasts Hdfa, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    normal human dermal fibroblast nhdf  (ATCC)


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    ATCC normal human dermal fibroblast nhdf
    A UMAP plot of 33,118 FBs coloured by cell subtype. Each dot denotes a single cell. B Heatmap showing differences in hallmark pathway activities scored per cell with GSVA among different <t>fibroblast</t> subtypes. ( C ) Dot plot of the expression levels of marker genes for FB subtypes defined in A . Dot size corresponds to the percent of expressing cells, and dot colour indicates the average expression levels. D UMAP plot of all fibroblast subtypes from the skin of the IMQ group and WT group, coloured by cell type. E Bulk RNA-seq and scRNA-seq analysis of the DEGs in the IMQ group and WT group (bulk RNA-seq data source GSM2299980 (WT), GSM2299981 (WT), GSM229998 (IMQ), GSM2299983 (IMQ)). F Violin plots showing the top DEGs between IMQ and WT in all fibroblast subtypes. G KEGG pathway map of IMQ vs. WT scRNA-seq in FBs. H Western blot image of Vim, Lcn2 and IL-17 expressions in HaCaT cells treated with IL-17 for indicated time. I – K Histogram of protein expression level in H . L – N Quantification of Vim, Lcn2 and IL-17 mRNA expressions in <t>NHDF</t> cells stimulated with IL-17 (20 ng/mL) for 6 h.
    Normal Human Dermal Fibroblast Nhdf, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Single-cell RNA-seq reveals keratinocyte and fibroblast heterogeneity and their crosstalk via epithelial-mesenchymal transition in psoriasis"

    Article Title: Single-cell RNA-seq reveals keratinocyte and fibroblast heterogeneity and their crosstalk via epithelial-mesenchymal transition in psoriasis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-024-06583-z

    A UMAP plot of 33,118 FBs coloured by cell subtype. Each dot denotes a single cell. B Heatmap showing differences in hallmark pathway activities scored per cell with GSVA among different fibroblast subtypes. ( C ) Dot plot of the expression levels of marker genes for FB subtypes defined in A . Dot size corresponds to the percent of expressing cells, and dot colour indicates the average expression levels. D UMAP plot of all fibroblast subtypes from the skin of the IMQ group and WT group, coloured by cell type. E Bulk RNA-seq and scRNA-seq analysis of the DEGs in the IMQ group and WT group (bulk RNA-seq data source GSM2299980 (WT), GSM2299981 (WT), GSM229998 (IMQ), GSM2299983 (IMQ)). F Violin plots showing the top DEGs between IMQ and WT in all fibroblast subtypes. G KEGG pathway map of IMQ vs. WT scRNA-seq in FBs. H Western blot image of Vim, Lcn2 and IL-17 expressions in HaCaT cells treated with IL-17 for indicated time. I – K Histogram of protein expression level in H . L – N Quantification of Vim, Lcn2 and IL-17 mRNA expressions in NHDF cells stimulated with IL-17 (20 ng/mL) for 6 h.
    Figure Legend Snippet: A UMAP plot of 33,118 FBs coloured by cell subtype. Each dot denotes a single cell. B Heatmap showing differences in hallmark pathway activities scored per cell with GSVA among different fibroblast subtypes. ( C ) Dot plot of the expression levels of marker genes for FB subtypes defined in A . Dot size corresponds to the percent of expressing cells, and dot colour indicates the average expression levels. D UMAP plot of all fibroblast subtypes from the skin of the IMQ group and WT group, coloured by cell type. E Bulk RNA-seq and scRNA-seq analysis of the DEGs in the IMQ group and WT group (bulk RNA-seq data source GSM2299980 (WT), GSM2299981 (WT), GSM229998 (IMQ), GSM2299983 (IMQ)). F Violin plots showing the top DEGs between IMQ and WT in all fibroblast subtypes. G KEGG pathway map of IMQ vs. WT scRNA-seq in FBs. H Western blot image of Vim, Lcn2 and IL-17 expressions in HaCaT cells treated with IL-17 for indicated time. I – K Histogram of protein expression level in H . L – N Quantification of Vim, Lcn2 and IL-17 mRNA expressions in NHDF cells stimulated with IL-17 (20 ng/mL) for 6 h.

    Techniques Used: Expressing, Marker, RNA Sequencing Assay, Western Blot

    normal human dermal fibroblasts nhdfs  (ATCC)


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    ATCC normal human dermal fibroblasts nhdfs
    Normal Human Dermal Fibroblasts Nhdfs, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human dermal fibroblasts  (ATCC)


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    ATCC human dermal fibroblasts
    Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC primary human dermal fibroblasts hdf
    ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal <t>fibroblasts</t> <t>(HDF)</t> were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.
    Primary Human Dermal Fibroblasts Hdf, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC normal human dermal nhd fibroblast cell lines
    (A) Schematic graph illustrating the timeline for the culture and TGF-β1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and Normal Human Dermal <t>(NHD)</t> <t>fibroblast</t> cells. (B) RUNX1 expression rate in samples treated with TGF-β1 (in red) vs. control for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the two SSc-isolated fibroblast lines at 12 hours after exposure vs. the baseline. (D) The pathway analysis of Reactome gene sets shows the biological pathways and processes that are significantly represented within top DEG genes of SSc-isolated fibroblast lines 12 hours after TGF-β1 treatment vs. the baseline. (E) Fold change expression of TGF-β1 and CBFB in TGF-β1-induced SSc fibroblasts treated with Ro5-3335 ( RUNX1 inhibitor), compared to control. (F) Proliferation curve of NHD fibroblasts in the presence and absence of Ro5-3335. (G–H) The 3D collagen contraction assays, fixed (G) and floating ( H ) models, of NHD fibroblasts treated with Ro5-3335. SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. (Student’s t-test P- value: **0.001–0.01, ****<0.0001 in GraphPad Prism v9)
    Normal Human Dermal Nhd Fibroblast Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC normal primary human dermal fibroblasts
    αSMA gene expression and protein levels are reduced in TGFβ1-activated <t>fibroblasts</t> following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.
    Normal Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC normal human ws1 dermal fibroblasts
    αSMA gene expression and protein levels are reduced in TGFβ1-activated <t>fibroblasts</t> following ketotifen treatment. ( A , B ) HDFa and ( C , D ) <t>WS1</t> fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.
    Normal Human Ws1 Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC primary human dermal fibroblasts
    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal <t>fibroblasts.</t> At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
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    ATCC primary human dermal fibroblasts phdf
    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal <t>fibroblasts.</t> At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
    Primary Human Dermal Fibroblasts Phdf, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human dermal fibroblasts hdfa
    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal <t>fibroblasts.</t> At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
    Human Dermal Fibroblasts Hdfa, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC normal human dermal fibroblast nhdf
    A UMAP plot of 33,118 FBs coloured by cell subtype. Each dot denotes a single cell. B Heatmap showing differences in hallmark pathway activities scored per cell with GSVA among different <t>fibroblast</t> subtypes. ( C ) Dot plot of the expression levels of marker genes for FB subtypes defined in A . Dot size corresponds to the percent of expressing cells, and dot colour indicates the average expression levels. D UMAP plot of all fibroblast subtypes from the skin of the IMQ group and WT group, coloured by cell type. E Bulk RNA-seq and scRNA-seq analysis of the DEGs in the IMQ group and WT group (bulk RNA-seq data source GSM2299980 (WT), GSM2299981 (WT), GSM229998 (IMQ), GSM2299983 (IMQ)). F Violin plots showing the top DEGs between IMQ and WT in all fibroblast subtypes. G KEGG pathway map of IMQ vs. WT scRNA-seq in FBs. H Western blot image of Vim, Lcn2 and IL-17 expressions in HaCaT cells treated with IL-17 for indicated time. I – K Histogram of protein expression level in H . L – N Quantification of Vim, Lcn2 and IL-17 mRNA expressions in <t>NHDF</t> cells stimulated with IL-17 (20 ng/mL) for 6 h.
    Normal Human Dermal Fibroblast Nhdf, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    ATCC normal human dermal fibroblasts nhdfs
    A UMAP plot of 33,118 FBs coloured by cell subtype. Each dot denotes a single cell. B Heatmap showing differences in hallmark pathway activities scored per cell with GSVA among different <t>fibroblast</t> subtypes. ( C ) Dot plot of the expression levels of marker genes for FB subtypes defined in A . Dot size corresponds to the percent of expressing cells, and dot colour indicates the average expression levels. D UMAP plot of all fibroblast subtypes from the skin of the IMQ group and WT group, coloured by cell type. E Bulk RNA-seq and scRNA-seq analysis of the DEGs in the IMQ group and WT group (bulk RNA-seq data source GSM2299980 (WT), GSM2299981 (WT), GSM229998 (IMQ), GSM2299983 (IMQ)). F Violin plots showing the top DEGs between IMQ and WT in all fibroblast subtypes. G KEGG pathway map of IMQ vs. WT scRNA-seq in FBs. H Western blot image of Vim, Lcn2 and IL-17 expressions in HaCaT cells treated with IL-17 for indicated time. I – K Histogram of protein expression level in H . L – N Quantification of Vim, Lcn2 and IL-17 mRNA expressions in <t>NHDF</t> cells stimulated with IL-17 (20 ng/mL) for 6 h.
    Normal Human Dermal Fibroblasts Nhdfs, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal human dermal fibroblasts nhdfs/product/ATCC
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    86
    ATCC human dermal fibroblasts
    A UMAP plot of 33,118 FBs coloured by cell subtype. Each dot denotes a single cell. B Heatmap showing differences in hallmark pathway activities scored per cell with GSVA among different <t>fibroblast</t> subtypes. ( C ) Dot plot of the expression levels of marker genes for FB subtypes defined in A . Dot size corresponds to the percent of expressing cells, and dot colour indicates the average expression levels. D UMAP plot of all fibroblast subtypes from the skin of the IMQ group and WT group, coloured by cell type. E Bulk RNA-seq and scRNA-seq analysis of the DEGs in the IMQ group and WT group (bulk RNA-seq data source GSM2299980 (WT), GSM2299981 (WT), GSM229998 (IMQ), GSM2299983 (IMQ)). F Violin plots showing the top DEGs between IMQ and WT in all fibroblast subtypes. G KEGG pathway map of IMQ vs. WT scRNA-seq in FBs. H Western blot image of Vim, Lcn2 and IL-17 expressions in HaCaT cells treated with IL-17 for indicated time. I – K Histogram of protein expression level in H . L – N Quantification of Vim, Lcn2 and IL-17 mRNA expressions in <t>NHDF</t> cells stimulated with IL-17 (20 ng/mL) for 6 h.
    Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human dermal fibroblasts/product/ATCC
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    Image Search Results


    ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.

    Journal: bioRxiv

    Article Title: Chloride Homeostasis Regulates cGAS-STING Signaling

    doi: 10.1101/2024.04.08.588475

    Figure Lengend Snippet: ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.

    Article Snippet: Primary Human Dermal Fibroblasts (HDF) were purchased from ATCC (Manassas, VA, USA).

    Techniques: Activation Assay, Cell Culture, Activity Assay, Transfection, Comparison, Expressing, Quantitative RT-PCR, Western Blot, Staining

    (A) Schematic graph illustrating the timeline for the culture and TGF-β1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and Normal Human Dermal (NHD) fibroblast cells. (B) RUNX1 expression rate in samples treated with TGF-β1 (in red) vs. control for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the two SSc-isolated fibroblast lines at 12 hours after exposure vs. the baseline. (D) The pathway analysis of Reactome gene sets shows the biological pathways and processes that are significantly represented within top DEG genes of SSc-isolated fibroblast lines 12 hours after TGF-β1 treatment vs. the baseline. (E) Fold change expression of TGF-β1 and CBFB in TGF-β1-induced SSc fibroblasts treated with Ro5-3335 ( RUNX1 inhibitor), compared to control. (F) Proliferation curve of NHD fibroblasts in the presence and absence of Ro5-3335. (G–H) The 3D collagen contraction assays, fixed (G) and floating ( H ) models, of NHD fibroblasts treated with Ro5-3335. SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. (Student’s t-test P- value: **0.001–0.01, ****<0.0001 in GraphPad Prism v9)

    Journal: bioRxiv

    Article Title: RUNX1 is Expressed in a Subpopulation of Dermal Fibroblasts and Higher RUNX1 Levels are Associated with the Severity of Systemic Sclerosis

    doi: 10.1101/2024.04.03.587966

    Figure Lengend Snippet: (A) Schematic graph illustrating the timeline for the culture and TGF-β1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and Normal Human Dermal (NHD) fibroblast cells. (B) RUNX1 expression rate in samples treated with TGF-β1 (in red) vs. control for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the two SSc-isolated fibroblast lines at 12 hours after exposure vs. the baseline. (D) The pathway analysis of Reactome gene sets shows the biological pathways and processes that are significantly represented within top DEG genes of SSc-isolated fibroblast lines 12 hours after TGF-β1 treatment vs. the baseline. (E) Fold change expression of TGF-β1 and CBFB in TGF-β1-induced SSc fibroblasts treated with Ro5-3335 ( RUNX1 inhibitor), compared to control. (F) Proliferation curve of NHD fibroblasts in the presence and absence of Ro5-3335. (G–H) The 3D collagen contraction assays, fixed (G) and floating ( H ) models, of NHD fibroblasts treated with Ro5-3335. SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. (Student’s t-test P- value: **0.001–0.01, ****<0.0001 in GraphPad Prism v9)

    Article Snippet: We analyzed a DNA microarray dataset previously generated by our lab, consisting of two independent SSc fibroblasts, one healthy control fibroblast isolated in parallel, and one normal human dermal (NHD) fibroblast cell lines obtained from ATCC.

    Techniques: Isolation, Expressing, Positive Control

    αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

    Ketotifen treatment reduced αSMA fibres in TGFβ1-activated fibroblasts. HDFa cells were cultured on poly- d -lysine-coated coverslips and treated for 48 h with media, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A ) Representative images of immunofluorescence staining (200X total magnification) probing for αSMA and DAPI are shown on the different groups of HDFa cells. ( B ) Quantification of αSMA+ staining (green) was plotted per DAPI+ cell (blue). Measurements for each biological replicate were averaged from five microscopic fields of view. Data shown as mean ± SEM. n = 3 per treatment condition. ** p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA, alpha-smooth muscle actin, DAPI, 4′, 6-diamidino-2-phenylindole, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta 1.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Ketotifen treatment reduced αSMA fibres in TGFβ1-activated fibroblasts. HDFa cells were cultured on poly- d -lysine-coated coverslips and treated for 48 h with media, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A ) Representative images of immunofluorescence staining (200X total magnification) probing for αSMA and DAPI are shown on the different groups of HDFa cells. ( B ) Quantification of αSMA+ staining (green) was plotted per DAPI+ cell (blue). Measurements for each biological replicate were averaged from five microscopic fields of view. Data shown as mean ± SEM. n = 3 per treatment condition. ** p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA, alpha-smooth muscle actin, DAPI, 4′, 6-diamidino-2-phenylindole, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta 1.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Cell Culture, Immunofluorescence, Staining

    Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Expressing

    Ketotifen treatment increased phosphorylation of YAP, decreased TAZ protein levels, and inhibited AKT phosphorylation. HDFa cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A , B ) Gene expression of YAP ( YAP1 ) and TAZ ( WWTR1 ) were assessed using RT-qPCR and normalized to housekeeping gene HPRT . ( C ) Phosphorylated YAP at serine residue 127 and total YAP protein were measured using western blot and semi-quantitative assessments made by normalization to GAPDH. ( D ) TAZ protein was measured and plotted in the same manner. ( E ) Representative images of the blots are shown. ( F ) Phosphorylated AKT at serine residue 473 was measured in TGFβ1-treated fibroblasts, in the presence of ketotifen or AKT inhibitor, MK2206 (left). Representative blot images of phosphorylated AKT are on the right. Full-length blots are available in Supplementary Fig. . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001. αSMA alpha-smooth muscle actin, AKT protein kinase B, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Ketotifen treatment increased phosphorylation of YAP, decreased TAZ protein levels, and inhibited AKT phosphorylation. HDFa cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A , B ) Gene expression of YAP ( YAP1 ) and TAZ ( WWTR1 ) were assessed using RT-qPCR and normalized to housekeeping gene HPRT . ( C ) Phosphorylated YAP at serine residue 127 and total YAP protein were measured using western blot and semi-quantitative assessments made by normalization to GAPDH. ( D ) TAZ protein was measured and plotted in the same manner. ( E ) Representative images of the blots are shown. ( F ) Phosphorylated AKT at serine residue 473 was measured in TGFβ1-treated fibroblasts, in the presence of ketotifen or AKT inhibitor, MK2206 (left). Representative blot images of phosphorylated AKT are on the right. Full-length blots are available in Supplementary Fig. . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001. αSMA alpha-smooth muscle actin, AKT protein kinase B, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Expressing, Quantitative RT-PCR, Residue, Western Blot, Binding Assay

    Ketotifen fumarate treatment impaired TGFβ1-stimulated fibroblast contraction in an in vitro model of fibroblast-populated gel contraction. ( A ) Representative images of fibroblast-populated collagen gels in DF10 (DMEM, with 10% FBS) culture media with or without ketotifen fumarate at 0- and 4 h post-treatment. ( B ) Contracture of activated fibroblast-populated collagen gels was determined and plotted over the course of 24 h as mean gel area in normal or ketotifen-supplemented media. ( C ) TGFβ1 or TGFβ1 and ketotifen fumarate-treated fibroblasts were seeded into collagen gels and contracture measured in normal culture media. ( D ) ACTA2 is significantly reduced in ketotifen-treated TGFβ1-activated fibroblasts compared to TGFβ1 treatment at 4 h. Gene expression data is normalized to housekeeping gene HPRT . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; *** p < 0.001; **** p < 0.0001. DF10 10% FBS-DMEM, DF10-K 10% FBS-DMEM with ketotifen fumarate, TGFβ1 transforming growth factor-beta 1, TK25 transforming growth factor-beta 1 with 25 μM ketotifen.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Ketotifen fumarate treatment impaired TGFβ1-stimulated fibroblast contraction in an in vitro model of fibroblast-populated gel contraction. ( A ) Representative images of fibroblast-populated collagen gels in DF10 (DMEM, with 10% FBS) culture media with or without ketotifen fumarate at 0- and 4 h post-treatment. ( B ) Contracture of activated fibroblast-populated collagen gels was determined and plotted over the course of 24 h as mean gel area in normal or ketotifen-supplemented media. ( C ) TGFβ1 or TGFβ1 and ketotifen fumarate-treated fibroblasts were seeded into collagen gels and contracture measured in normal culture media. ( D ) ACTA2 is significantly reduced in ketotifen-treated TGFβ1-activated fibroblasts compared to TGFβ1 treatment at 4 h. Gene expression data is normalized to housekeeping gene HPRT . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; *** p < 0.001; **** p < 0.0001. DF10 10% FBS-DMEM, DF10-K 10% FBS-DMEM with ketotifen fumarate, TGFβ1 transforming growth factor-beta 1, TK25 transforming growth factor-beta 1 with 25 μM ketotifen.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: In Vitro, Expressing

    Schematic of the effects of ketotifen fumarate on fibroblast phenotype and associated cellular mechanisms. ( A ) Ketotifen fumarate inhibits the effects of TGFβ1 on fibroblasts, decreasing αSMA gene and protein levels, cytoskeletal-associated genes, and contractility. ( B ) Ketotifen fumarate treatment reduces ACTA2 , CNN1 , and TAGLN , and decreases TAZ gene and protein levels. Increased phosphorylation of YAP at serine residue 127 is also seen, promoting cytosolic localization. Ketotifen also affects the PI3K-AKT pathway by reducing phosphorylation of AKT at serine residue 473. AKT protein kinase B, αSMA alpha-smooth muscle actin, CNN1 calponin 1, PI3K phosphatidylinositol-3-kinase, TAGLN transgelin, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Schematic of the effects of ketotifen fumarate on fibroblast phenotype and associated cellular mechanisms. ( A ) Ketotifen fumarate inhibits the effects of TGFβ1 on fibroblasts, decreasing αSMA gene and protein levels, cytoskeletal-associated genes, and contractility. ( B ) Ketotifen fumarate treatment reduces ACTA2 , CNN1 , and TAGLN , and decreases TAZ gene and protein levels. Increased phosphorylation of YAP at serine residue 127 is also seen, promoting cytosolic localization. Ketotifen also affects the PI3K-AKT pathway by reducing phosphorylation of AKT at serine residue 473. AKT protein kinase B, αSMA alpha-smooth muscle actin, CNN1 calponin 1, PI3K phosphatidylinositol-3-kinase, TAGLN transgelin, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Residue, Binding Assay

    αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

    Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Expressing

    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Journal: Scientific Reports

    Article Title: Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors

    doi: 10.1038/s41598-024-54941-w

    Figure Lengend Snippet: HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Article Snippet: Primary human dermal fibroblasts were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured using a fibroblast growth medium purchased from the manufacturer.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for VEGF, PGF, or ANGPT1, expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Journal: Scientific Reports

    Article Title: Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors

    doi: 10.1038/s41598-024-54941-w

    Figure Lengend Snippet: HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for VEGF, PGF, or ANGPT1, expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Article Snippet: Primary human dermal fibroblasts were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured using a fibroblast growth medium purchased from the manufacturer.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for FLT-1, or EDN1 expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Journal: Scientific Reports

    Article Title: Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors

    doi: 10.1038/s41598-024-54941-w

    Figure Lengend Snippet: HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for FLT-1, or EDN1 expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Article Snippet: Primary human dermal fibroblasts were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured using a fibroblast growth medium purchased from the manufacturer.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    A UMAP plot of 33,118 FBs coloured by cell subtype. Each dot denotes a single cell. B Heatmap showing differences in hallmark pathway activities scored per cell with GSVA among different fibroblast subtypes. ( C ) Dot plot of the expression levels of marker genes for FB subtypes defined in A . Dot size corresponds to the percent of expressing cells, and dot colour indicates the average expression levels. D UMAP plot of all fibroblast subtypes from the skin of the IMQ group and WT group, coloured by cell type. E Bulk RNA-seq and scRNA-seq analysis of the DEGs in the IMQ group and WT group (bulk RNA-seq data source GSM2299980 (WT), GSM2299981 (WT), GSM229998 (IMQ), GSM2299983 (IMQ)). F Violin plots showing the top DEGs between IMQ and WT in all fibroblast subtypes. G KEGG pathway map of IMQ vs. WT scRNA-seq in FBs. H Western blot image of Vim, Lcn2 and IL-17 expressions in HaCaT cells treated with IL-17 for indicated time. I – K Histogram of protein expression level in H . L – N Quantification of Vim, Lcn2 and IL-17 mRNA expressions in NHDF cells stimulated with IL-17 (20 ng/mL) for 6 h.

    Journal: Cell Death & Disease

    Article Title: Single-cell RNA-seq reveals keratinocyte and fibroblast heterogeneity and their crosstalk via epithelial-mesenchymal transition in psoriasis

    doi: 10.1038/s41419-024-06583-z

    Figure Lengend Snippet: A UMAP plot of 33,118 FBs coloured by cell subtype. Each dot denotes a single cell. B Heatmap showing differences in hallmark pathway activities scored per cell with GSVA among different fibroblast subtypes. ( C ) Dot plot of the expression levels of marker genes for FB subtypes defined in A . Dot size corresponds to the percent of expressing cells, and dot colour indicates the average expression levels. D UMAP plot of all fibroblast subtypes from the skin of the IMQ group and WT group, coloured by cell type. E Bulk RNA-seq and scRNA-seq analysis of the DEGs in the IMQ group and WT group (bulk RNA-seq data source GSM2299980 (WT), GSM2299981 (WT), GSM229998 (IMQ), GSM2299983 (IMQ)). F Violin plots showing the top DEGs between IMQ and WT in all fibroblast subtypes. G KEGG pathway map of IMQ vs. WT scRNA-seq in FBs. H Western blot image of Vim, Lcn2 and IL-17 expressions in HaCaT cells treated with IL-17 for indicated time. I – K Histogram of protein expression level in H . L – N Quantification of Vim, Lcn2 and IL-17 mRNA expressions in NHDF cells stimulated with IL-17 (20 ng/mL) for 6 h.

    Article Snippet: Normal human dermal fibroblast (NHDF) was cultured and expanded in Fibroblast Basal Medium (PCS-201-030, ATCC, U.S.A) supplemented with Fibroblast Growth Kit-Low serum (PCS-201-041, ATCC, U.S.A) and 1% penicillin-streptomycin.

    Techniques: Expressing, Marker, RNA Sequencing Assay, Western Blot