human keratinocyte growth factor  (R&D Systems)

Journal: STAR Protocols

Article Title: Protocol for differentiation of human embryonic stem cells into surface epithelium and functional keratinocytes

doi: 10.1016/j.xpro.2025.103919

Figure Lengend Snippet: Induction of SE cells into mature keratinocyte progenitors (A) Left panel: Phase contrast images of the induced keratinocyte progenitors after 45 days of differentiation and 4 days after passaging. Scale bar: 100 μm. Right panel: Immunostaining for KRT14 and TP63 in the induced keratinocyte progenitors. Scale bar: 100 μm. (B) qRT-PCR analysis of representative genes in SE and induced keratinocyte progenitors. qRT-PCR values were normalized to the SE group. Values are presented as means ± SD (n=3 biological replicates; ∗∗P<0.01; ∗∗∗P<0.001, t test).

Article Snippet: Keratinocyte growth factor (KGF) , MedChemExpress , Cat#HY- P70673.

Techniques: Passaging, Immunostaining, Quantitative RT-PCR

Journal: STAR Protocols

Article Title: Protocol for differentiation of human embryonic stem cells into surface epithelium and functional keratinocytes

doi: 10.1016/j.xpro.2025.103919

Figure Lengend Snippet: Terminal differentiation of keratinocyte progenitors (A) Immunostaining for KRT1 and KRT10 in the keratinocytes after 7 days of CaCl 2 treatment. Scale bar: 100 μm. (B) qRT-PCR analysis of representative genes in keratinocyte progenitors and keratinocytes after 7 days of CaCl 2 treatment. qRT-PCR values were normalized to the keratinocyte progenitor group. Values are presented as means ± SD (n=3 biological replicates; ∗P<0.05; ∗∗P<0.01, t test).

Article Snippet: Keratinocyte growth factor (KGF) , MedChemExpress , Cat#HY- P70673.

Techniques: Immunostaining, Quantitative RT-PCR

Journal: STAR Protocols

Article Title: Protocol for differentiation of human embryonic stem cells into surface epithelium and functional keratinocytes

doi: 10.1016/j.xpro.2025.103919

Figure Lengend Snippet: Air-lifting assay of keratinocyte progenitors (A) Schematic diagram illustrating the procedure of the air-lifting culture assay. (B) Immunostaining for KRT1, KRT10 and TP63 in the stratified keratinocytes after 10 days of air-lifting induction. Scale bar: 100 μm.

Article Snippet: Keratinocyte growth factor (KGF) , MedChemExpress , Cat#HY- P70673.

Techniques: Immunostaining

Journal: Advanced Science

Article Title: Reprogramming of Fatty Acid Metabolism via PPARα‐Orchestrated FADS2 in Keratinocytes Modulates Skin Inflammation in Psoriasis

doi: 10.1002/advs.202417049

Figure Lengend Snippet: FADS2 is downregulated in keratinocytes of psoriatic lesions. A) Schematic illustration of the desaturation pathway of linoleic acid (omega‐6 [n‐6], red) and alpha‐linolenic acid (omega‐3 [n‐3], blue), catalyzed by key enzymes to generate long‐chain polyunsaturated fatty acids (LC‐PUFAs). B) Transcriptomic analysis of FADS2, FADS1 , and ELOVL5 expression in lesional (PP) and non‐lesional (PN) skin of psoriasis patients (n = 58) and in normal skin from healthy controls (NN, n = 64) based on the GEO dataset GSE13355 . C) Transcriptomic analysis of FADS2, FADS1 , and ELOVL5 expression in baseline lesional (B‐LS) and non‐lesional (B‐NL) skin of psoriasis patients (n = 59), and in lesional skin after 12 weeks of guselkumab treatment (P‐LS), from GEO dataset GSE51440 . D) RT‐qPCR validation of FADS2, FADS1 , and ELOVL5 expression in normal skin from healthy controls (NN) (n = 4) and lesional skin tissue from psoriasis patients (PS) (n = 5). E–G) Representative immunofluorescence images of FADS2 (E), FADS1 (F), and ELOVL5 (G) staining, along with the keratinocyte marker K14, in normal skin from healthy controls and lesional skin tissue from psoriasis patients. Dashed line indicates the border between the epidermis and dermis. H) Representative immunofluorescence images of FADS2 and K14 co‐staining in lesional skin tissue from psoriasis patients at baseline and after 12 weeks guselkumab treatment. I) Representative immunofluorescence images of FADS2 and K14 co‐staining in healthy skin from control mice (Ctrl) and imiquimod (IMQ)‐induced psoriatic skin lesions. Scale bar, 50 µm. Data are presented as mean ± SD. Statistical significance was determined by one‐way ANOVA (B,C) or unpaired two‐tailed Student's t ‐test (D). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: To verify whether FADS2 silencing promotes inflammation through the NF‐κB signaling pathway, HaCaT cells were transfected with si FADS2 or siNC for 24 h followed by treatment with the NF‐κB inhibitor BAY 11–7082 (5 μм; #HY‐13453, MedChemExpress) or DMSO for 2 h before M5 stimulation for 12 h. To investigate the anti‐inflammatory effect of DHA on keratinocyte, HaCaT cells were pretreated with 100 μ m DHA (#HY‐B2167, Medchemexpress) or vehicle dissolved in ethanol and bound to fatty acid‐free bovine serum albumin in complete growth medium for 36 h and then stimulated with 10 ng mL −1 M5 for another 12 h. To investigate whether DHA supplementation could reverse the enhanced inflammatory response caused by FADS2 knockdown, HaCaT cells were transfected with si FADS2 or siNC for 12 h, followed by treatment with 100 μ m DHA for 12 h prior to M5 stimulation for an additional 12 h. To explore the effect of PPARα on keratinocyte inflammation, HaCaT cells were pretreated with 50 μ m WY14643 (HY‐16995, Medchemexpress) or DMSO for 21 h, followed by M5 for 3 h. To assess whether PPARα modulates inflammation through FADS2 regulation, HaCaT cells were transfected with si FADS2 or siNC for 24 h, followed by treatment with WY14643 or vehicle for 4 h prior to 12 h M5 stimulation.

Techniques: Expressing, Quantitative RT-PCR, Biomarker Discovery, Immunofluorescence, Staining, Marker, Control, Two Tailed Test

Journal: Advanced Science

Article Title: Reprogramming of Fatty Acid Metabolism via PPARα‐Orchestrated FADS2 in Keratinocytes Modulates Skin Inflammation in Psoriasis

doi: 10.1002/advs.202417049

Figure Lengend Snippet: FADS2 modulates psoriatic inflammation in keratinocytes through NF‐κB activation. A) RT‐qPCR analysis of the indicated genes in HaCaT cells transfected with FADS2 siRNA (si FADS2 ) or control siRNA (siNC) for 24 h, followed by stimulation with PBS or a cytokine cocktail (M5) for 12 h (n = 3). B) ELISA quantification of CXCL1 and CXCL8 protein levels in both cell lysates and supernatants of HaCaT cells treated as (A) (n = 3). C) Top 10 enriched Gene Ontology (GO) molecular function terms from RNA‐sequencing (RNA‐seq) analysis of differential expression genes (DEGs) in FADS2 ‐silenced (si FADS2 ) versus control (siNC) HaCaT cells after M5 stimulation. DEGs were defined by |fold change| >1.5 & adjusted P <0.05. D) Gene set enrichment analysis (GSEA) of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment from RNA‐seq results in FADS2 ‐silenced (si FADS2 ) versus control (siNC) HaCaT cells after M5 stimulation. E) Representative immunofluorescence images of phosphorylated NF‐κB p65 (pNF‐κB) and K14 co‐staining in normal skin from healthy controls and lesional skin tissue from psoriasis patients. F) Immunoblotting of pNF‐κB and total‐NF‐κB p65 (NF‐κB) in HaCaT cells transfected with si FADS2 or siNC for 24 h and stimulated with PBS or M5 for 1 h. G) RT‐qPCR analysis of the indicated genes in HaCaT cells transfected with si FADS2 or siNC and stimulated with M5 after BAY 11–7082 or DMSO pretreatment (n = 3). H) RT‐qPCR analysis of FADS2 and the indicated genes in HaCaT cells transfected with FADS2 overexpression plasmids ( FADS2 OE) or empty vector for 48 h followed by PBS or M5 treatment for 10 h (n = 3). Scale bar, 50 µm. Data are presented as mean ± SD. Statistical significance was determined by two‐way ANOVA (A,G,H) or unpaired two‐tailed Student's t ‐test (B, H). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: To verify whether FADS2 silencing promotes inflammation through the NF‐κB signaling pathway, HaCaT cells were transfected with si FADS2 or siNC for 24 h followed by treatment with the NF‐κB inhibitor BAY 11–7082 (5 μм; #HY‐13453, MedChemExpress) or DMSO for 2 h before M5 stimulation for 12 h. To investigate the anti‐inflammatory effect of DHA on keratinocyte, HaCaT cells were pretreated with 100 μ m DHA (#HY‐B2167, Medchemexpress) or vehicle dissolved in ethanol and bound to fatty acid‐free bovine serum albumin in complete growth medium for 36 h and then stimulated with 10 ng mL −1 M5 for another 12 h. To investigate whether DHA supplementation could reverse the enhanced inflammatory response caused by FADS2 knockdown, HaCaT cells were transfected with si FADS2 or siNC for 12 h, followed by treatment with 100 μ m DHA for 12 h prior to M5 stimulation for an additional 12 h. To explore the effect of PPARα on keratinocyte inflammation, HaCaT cells were pretreated with 50 μ m WY14643 (HY‐16995, Medchemexpress) or DMSO for 21 h, followed by M5 for 3 h. To assess whether PPARα modulates inflammation through FADS2 regulation, HaCaT cells were transfected with si FADS2 or siNC for 24 h, followed by treatment with WY14643 or vehicle for 4 h prior to 12 h M5 stimulation.

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Control, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Quantitative Proteomics, Immunofluorescence, Staining, Western Blot, Over Expression, Plasmid Preparation, Two Tailed Test

Journal: Advanced Science

Article Title: Reprogramming of Fatty Acid Metabolism via PPARα‐Orchestrated FADS2 in Keratinocytes Modulates Skin Inflammation in Psoriasis

doi: 10.1002/advs.202417049

Figure Lengend Snippet: Keratinocyte‐specific modulation of FADS2 expression by AAV9‐K14 delivery system alters IMQ‐induced skin inflammation. A‐I) BALB/c mice were subjected to IMQ‐induced psoriasis‐like dermatitis on the dorsal skin three weeks after intradermal injection of AAV9‐K14‐sh Fads2 or AAV9‐K14‐shNC. A) Representative phenotypic images and H&E staining images of IMQ‐induced skin lesions treated as (A‐I). B,C) Quantification of PASI scores (B) and epidermal thickness (C) of IMQ‐induced skin lesions treated as (A‐I) (n = 6). D,E) Representative immunofluorescence images of Ki67 staining (D) and quantitation of Ki67 + epidermal cells (E) in IMQ‐induced skin lesions treated as (A‐I) in the indicated locations (n = 4). F) RT‐qPCR analysis of the indicated genes in IMQ‐induced skin lesions treated as (A‐I) (n = 6). G,H) Representative flow cytometry plot (G) and quantification (H) of neutrophils in IMQ‐induced skin lesions treated as (A‐I) (n = 4). I) Representative immunofluorescence images of pNF‐κB and K14 co‐staining in IMQ‐induced skin lesions treated as (A‐I). J‐R) BALB/c mice were subjected to IMQ‐induced psoriasis‐like dermatitis on the dorsal skin three weeks after intradermal injection of AAV9‐K14‐flag‐ Fads2 (AAV9‐K14‐ Fads2 ) or AAV9‐K14‐NC (AAV9‐K14‐Ctrl). J) Representative phenotypic images and H&E staining images of IMQ‐induced skin lesions treated as (J–R). K,L) Quantification of PASI scores (K), epidermal thickness (L) in IMQ‐induced skin lesions treated as (J–R) (n = 6). M,N) Representative immunofluorescence images of Ki67 staining (M) and quantitation of Ki67 + epidermal cells (N) in IMQ‐induced skin lesions treated as (J‐R) (n = 4). O) RT‐qPCR analysis of the indicated genes in IMQ‐induced skin lesions treated as (J–R) (n = 6). P,Q) Representative flow cytometry plot (P) and quantification (Q) of neutrophils in IMQ‐induced skin lesions treated as (J–R) (n = 6). R) Representative immunofluorescence images of pNF‐κB and K14 co‐staining in IMQ‐induced skin lesions treated as (J–R). Scale bar, 50 µm. Data are presented as mean ± SD. Statistical significance was determined by unpaired two‐tailed Student's t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: To verify whether FADS2 silencing promotes inflammation through the NF‐κB signaling pathway, HaCaT cells were transfected with si FADS2 or siNC for 24 h followed by treatment with the NF‐κB inhibitor BAY 11–7082 (5 μм; #HY‐13453, MedChemExpress) or DMSO for 2 h before M5 stimulation for 12 h. To investigate the anti‐inflammatory effect of DHA on keratinocyte, HaCaT cells were pretreated with 100 μ m DHA (#HY‐B2167, Medchemexpress) or vehicle dissolved in ethanol and bound to fatty acid‐free bovine serum albumin in complete growth medium for 36 h and then stimulated with 10 ng mL −1 M5 for another 12 h. To investigate whether DHA supplementation could reverse the enhanced inflammatory response caused by FADS2 knockdown, HaCaT cells were transfected with si FADS2 or siNC for 12 h, followed by treatment with 100 μ m DHA for 12 h prior to M5 stimulation for an additional 12 h. To explore the effect of PPARα on keratinocyte inflammation, HaCaT cells were pretreated with 50 μ m WY14643 (HY‐16995, Medchemexpress) or DMSO for 21 h, followed by M5 for 3 h. To assess whether PPARα modulates inflammation through FADS2 regulation, HaCaT cells were transfected with si FADS2 or siNC for 24 h, followed by treatment with WY14643 or vehicle for 4 h prior to 12 h M5 stimulation.

Techniques: Expressing, Injection, Staining, Immunofluorescence, Quantitation Assay, Quantitative RT-PCR, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: Reprogramming of Fatty Acid Metabolism via PPARα‐Orchestrated FADS2 in Keratinocytes Modulates Skin Inflammation in Psoriasis

doi: 10.1002/advs.202417049

Figure Lengend Snippet: Decreased FADS2 disrupts PUFA metabolism that orchestrates psoriasiform inflammation in keratinocytes. A) PUFA ratios from LC‐MS/MS‐based lipidomic analysis showing desaturation indexes related to FADS2 in HaCaT cells transfected with FADS2 siRNA and control siRNA after M5 stimulation (n = 3). B) RT‐qPCR analysis of the indicated genes in HaCaT cells treated with DHA or vehicle for 24 h and stimulated with PBS or M5 for 12 h (n = 3). C) Immunoblotting of pNF‐κB and total‐NF‐κB in HaCaT cells treated with DHA or vehicle for 24 h and stimulated with M5 for 1 h. D) RT‐qPCR analysis of the indicated genes in HaCaT cells transfected with si FADS2 or siNC and stimulated with M5 after DHA or vehicle pretreatment (n = 3). Data are presented as mean ± SD. Statistical significance was determined by unpaired two‐tailed Student's t test (A) or two‐way ANOVA (B,D). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: To verify whether FADS2 silencing promotes inflammation through the NF‐κB signaling pathway, HaCaT cells were transfected with si FADS2 or siNC for 24 h followed by treatment with the NF‐κB inhibitor BAY 11–7082 (5 μм; #HY‐13453, MedChemExpress) or DMSO for 2 h before M5 stimulation for 12 h. To investigate the anti‐inflammatory effect of DHA on keratinocyte, HaCaT cells were pretreated with 100 μ m DHA (#HY‐B2167, Medchemexpress) or vehicle dissolved in ethanol and bound to fatty acid‐free bovine serum albumin in complete growth medium for 36 h and then stimulated with 10 ng mL −1 M5 for another 12 h. To investigate whether DHA supplementation could reverse the enhanced inflammatory response caused by FADS2 knockdown, HaCaT cells were transfected with si FADS2 or siNC for 12 h, followed by treatment with 100 μ m DHA for 12 h prior to M5 stimulation for an additional 12 h. To explore the effect of PPARα on keratinocyte inflammation, HaCaT cells were pretreated with 50 μ m WY14643 (HY‐16995, Medchemexpress) or DMSO for 21 h, followed by M5 for 3 h. To assess whether PPARα modulates inflammation through FADS2 regulation, HaCaT cells were transfected with si FADS2 or siNC for 24 h, followed by treatment with WY14643 or vehicle for 4 h prior to 12 h M5 stimulation.

Techniques: Liquid Chromatography with Mass Spectroscopy, Transfection, Control, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Advanced Science

Article Title: Reprogramming of Fatty Acid Metabolism via PPARα‐Orchestrated FADS2 in Keratinocytes Modulates Skin Inflammation in Psoriasis

doi: 10.1002/advs.202417049

Figure Lengend Snippet: PPARα acts as an upstream positive regulator of FADS2 in psoriatic keratinocytes. A) RT‐qPCR analysis of PPARA expression in normal skin from healthy controls (NN, n = 4) and lesional skin tissue from psoriasis patients (PS, n = 5). B) Representative immunofluorescence images of PPARα and K14 co‐staining in normal skin from healthy controls and lesional skin tissue from psoriasis patients. C) RT‐qPCR analysis of PPARA in psoriatic lesional skin before and 10 weeks after infliximab treatment (n = 5). D) Representative images of PPARα immunofluorescence staining in lesional skin from psoriasis patients at baseline and week 10 following infliximab treatment. E) Representative immunofluorescence images of PPARα staining in healthy skin from control mice and skin lesions from IMQ‐induced psoriasis mouse model. F,G) RT‐qPCR analysis of PPARA (F) and FADS2 (G) expression in HaCaT cells stimulated with M5 for 12 h (n = 3). H) Immunoblotting of PPARα and FADS2 in HaCaT cells stimulated with M5 cytokines for the indicated time. I) RT‐qPCR analysis of PPARA , FADS2 , and indicated genes in HaCaT cells transfected with PPARA siRNA (si PPARA ) and control siRNA (siNC) for 24 h, followed by PBS or M5 stimulation for 12 h (n = 3). J) RT‐qPCR analysis of FADS2 and indicated genes in HaCaT cells pretreated with WY14643 or DMSO for 21 h, followed by PBS or M5 stimulation for 3 h (n = 3). K) ELISA quantification of CXCL1, CXCL8, and CSF3 protein levels in cell lysates and supernatants from HaCaT cells treated as in (J) (n = 3). L) RT‐qPCR analysis of the indicated genes in HaCaT cells transfected with si FADS2 and siNC for 24 h, followed by M5 stimulation for 3 h before WY14643 or DMSO pretreatment (n = 3). Scale bar, 50 µm. Data are presented as mean ± SD. Statistical significance was determined by unpaired two‐tailed Student's t test (A,F,G,K), paired two‐tailed Student's t test (C), or one‐way ANOVA (I,J,L). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: To verify whether FADS2 silencing promotes inflammation through the NF‐κB signaling pathway, HaCaT cells were transfected with si FADS2 or siNC for 24 h followed by treatment with the NF‐κB inhibitor BAY 11–7082 (5 μм; #HY‐13453, MedChemExpress) or DMSO for 2 h before M5 stimulation for 12 h. To investigate the anti‐inflammatory effect of DHA on keratinocyte, HaCaT cells were pretreated with 100 μ m DHA (#HY‐B2167, Medchemexpress) or vehicle dissolved in ethanol and bound to fatty acid‐free bovine serum albumin in complete growth medium for 36 h and then stimulated with 10 ng mL −1 M5 for another 12 h. To investigate whether DHA supplementation could reverse the enhanced inflammatory response caused by FADS2 knockdown, HaCaT cells were transfected with si FADS2 or siNC for 12 h, followed by treatment with 100 μ m DHA for 12 h prior to M5 stimulation for an additional 12 h. To explore the effect of PPARα on keratinocyte inflammation, HaCaT cells were pretreated with 50 μ m WY14643 (HY‐16995, Medchemexpress) or DMSO for 21 h, followed by M5 for 3 h. To assess whether PPARα modulates inflammation through FADS2 regulation, HaCaT cells were transfected with si FADS2 or siNC for 24 h, followed by treatment with WY14643 or vehicle for 4 h prior to 12 h M5 stimulation.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Control, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Reprogramming of Fatty Acid Metabolism via PPARα‐Orchestrated FADS2 in Keratinocytes Modulates Skin Inflammation in Psoriasis

doi: 10.1002/advs.202417049

Figure Lengend Snippet: The PPARα‐FADS2‐PUFA axis regulates psoriasiform inflammation in keratinocytes. PPARα positively regulates FADS2 expression and thus promotes the desaturation of PUFAs. In psoriatic keratinocytes, reduced PPARα expression leads to downregulation of FADS2, resulting in decreased synthesis of DHA. This reduction enhances NF‐κB phosphorylation and subsequently upregulates psoriasis‐associated inflammatory cytokines and chemokines. These chemokines further promote neutrophil recruitment into skin lesions, aggravating psoriasis‐like inflammation. This figure was created with BioRender.com. Reproduced with permission. Copyright 2025, https://BioRender.com/rnyxsoi .

Article Snippet: To verify whether FADS2 silencing promotes inflammation through the NF‐κB signaling pathway, HaCaT cells were transfected with si FADS2 or siNC for 24 h followed by treatment with the NF‐κB inhibitor BAY 11–7082 (5 μм; #HY‐13453, MedChemExpress) or DMSO for 2 h before M5 stimulation for 12 h. To investigate the anti‐inflammatory effect of DHA on keratinocyte, HaCaT cells were pretreated with 100 μ m DHA (#HY‐B2167, Medchemexpress) or vehicle dissolved in ethanol and bound to fatty acid‐free bovine serum albumin in complete growth medium for 36 h and then stimulated with 10 ng mL −1 M5 for another 12 h. To investigate whether DHA supplementation could reverse the enhanced inflammatory response caused by FADS2 knockdown, HaCaT cells were transfected with si FADS2 or siNC for 12 h, followed by treatment with 100 μ m DHA for 12 h prior to M5 stimulation for an additional 12 h. To explore the effect of PPARα on keratinocyte inflammation, HaCaT cells were pretreated with 50 μ m WY14643 (HY‐16995, Medchemexpress) or DMSO for 21 h, followed by M5 for 3 h. To assess whether PPARα modulates inflammation through FADS2 regulation, HaCaT cells were transfected with si FADS2 or siNC for 24 h, followed by treatment with WY14643 or vehicle for 4 h prior to 12 h M5 stimulation.

Techniques: Expressing, Phospho-proteomics