Journal: Redox Biology

Article Title: Macrophage AMPK activated by oxidative stress drives profibrotic crosstalk with tubular cells to accelerate renal fibrosis after ischemic and reperfusion injury

doi: 10.1016/j.redox.2025.104002

Figure Lengend Snippet: Arg1 + MMP12 + macrophage-derived TWEAK licenses PDGFB production in fibroTECs via Fn14 signaling. (A) Ligand-receptor communication analysis between different cell types in IRI kidneys. (B) The major incoming signaling of different cell types. (C) Bubble plots showing the expression of specific ligand-receptor pairs in the interaction between myeloid cell subsets and TEC subsets. (D) Bubble plots showing the expression of specific ligand-receptor pairs in the interaction between TEC subsets and myeloid cell subsets. (E, F) WT/AMPKα1 −/− BMDMs were treated with IL-4 and IL-13, and the expression levels of Arg 1 , Mmp12 , and Tnfsf12 were analyzed by qPCR (n = 5). (G, H) TCMK-1 cells were treated with CM from IL-4 and IL-13-stimulated WT/AMPKα1 −/− BMDMs. The expression of Pdgfb , Cxcl1 , and Egfr in TCMK-1 cells was analyzed by qPCR (n = 5). (I, J) TCMK-1 cells were treated with CM from IL-4 and IL-13-stimulated WT/AMPKα1 −/− BMDMs or together with recombinant TWEAK (rTWEAK). The expression of Pdgfb in TCMK-1 cells was analyzed by qPCR (n = 5). (K, L) TCMK-1 cells were treated with rTWEAK or rTWEAK and an Fn14 inhibitor. The expression of Pdgfb in TCMK-1 cells was detected by qPCR (n = 5). (M) Immunoblots and quantification of IKK-β and nuclear NF-κB p65 in TCMK-1 cells treated with PBS or rTWEAK (n = 3). The results represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: To examine the role of the TWEAK-Fn14 axis, cells were treated with recombinant TWEAK (MCE, HY-P7309, 100 ng/ml) and the Fn14 inhibitor, L524-0366 (Sigma, 509374, 10 μM).

Techniques: Derivative Assay, Expressing, Recombinant, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Effects of TWEAK on the proliferation and apoptosis of PDLSCs. (A) Line chart depicting the effects of various concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml) on PDLSC proliferation over 5 days, as assessed using the CCK-8 assay. (B) Statistical analysis of the CCK-8 data from day 5 of TWEAK stimulation shown in (A). (C) Statistical analysis of the average fluorescence intensity of TUNEL (green) following treatment with varying concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml). (D) TUNEL assay detecting the effects of various concentrations (0, 1, 5, 20, 50 and 100 ng/ml) of TWEAK on PDLSC apoptosis. Scale bar, 200 μ m. Statistical analysis was performed using a one-way ANOVA. * P<0.05; **** P<0.0001. Data are presented as the mean ± SD (n=5 or 6). CCK-8, Cell Counting Kit-8; OD450, optical density at 450 nm; PDLSC, periodontal ligament stem cell; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Article Snippet: Recombinant human cynomolgus TWEAK/TNF superfamily member 12 (TNFSF12) protein (mFc tag) (cat. no. 90094-C04H; Sino Biological, Inc.) was dissolved in sterile water to obtain a stock solution concentration of 50 μ g/ml.

Techniques: CCK-8 Assay, Fluorescence, TUNEL Assay, Cell Counting

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Effects of TWEAK on the migration and osteogenic differentiation of PDLSCs. (A) Effects of various concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml) on the number of migrating PDLSCs, and (B) quantitative analysis of the number of migrating cells (n=6). Scale bar, 400 μ m. (C) Effects of various concentrations of TWEAK on PDLSC migration toward scratch wounds over 24 h, and (D) quantitative analysis of the percentage of wound area reduction (n=12). Scale bar, 1 mm. (E) Effects of various concentrations of TWEAK on ALP staining in PDLSCs, and (F) quantitative analysis of grayscale values from ALP staining (n=6). Scale bar, 200 μ m. (G) Alizarin Red staining revealed the effects of various concentrations of TWEAK on PDLSC mineralization, and (H) quantitative analysis of grayscale values from Alizarin Red staining (n=6). Scale bar, 200 μ m. (I) Reverse transcription-quantitative PCR was used to assess the mRNA expression levels of RUNX2 , SP7 , ALP and OPG in PDLSCs after TWEAK stimulation (n=4), with β-actin serving as the internal control. (J) Western blot analysis of RUNX2, SP7, ALP and OPG protein expression in PDLSCs after TWEAK induction, and (K) semi-quantitative analysis of the gel band intensity, using β-actin as the internal control. Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. ALP, alkaline phosphatase; IOD, integral optical density; ns, not significant; OPG, osteoprotegerin; PDLSC, periodontal ligament stem cell; RUNX2, runt-related transcription factor 2; SP7, Sp7 transcription factor; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Article Snippet: Recombinant human cynomolgus TWEAK/TNF superfamily member 12 (TNFSF12) protein (mFc tag) (cat. no. 90094-C04H; Sino Biological, Inc.) was dissolved in sterile water to obtain a stock solution concentration of 50 μ g/ml.

Techniques: Migration, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Transcriptome analysis of the effect of TWEAK treatment on PDLSCs. (A) Venn diagram illustrating differentially expressed genes in PDLSCs following TWEAK treatment. (B) Multipoint differential scatter plot showing differentially expressed genes in PDLSCs following TWEAK treatment. (C) GO enrichment analysis comparing PDLSCs and PDLSCs treated with 50 ng/ml TWEAK. (D) KEGG enrichment analysis comparing PDLSCs and PDLSCs treated with 50 ng/ml TWEAK. (E) GO enrichment analysis comparing PDLSCs and PDLSCs treated with 100 ng/ml TWEAK. (F) KEGG enrichment analysis comparing PDLSCs and PDLSCs treated with 100 ng/ml TWEAK. (G) Western blot analysis was conducted to detect the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs stimulated with 50 and 100 ng/ml TWEAK. (H) Statistical analysis of protein band intensities from (G) (n=3). (I) Western blot analysis of the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs after Fn14 was silenced using an shRNA. (J) Statistical analysis of protein band intensities from (I) (n=3). Statistical analysis was performed using (G and H) one-way ANOVA or (I and J) a two-tailed Student's t-test. * P<0.05; ** P<0.01; *** P<0.001. Data are presented as the mean ± SD. FC, fold change; Fn14, fibroblast growth factor-inducible 14; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ns, not significant; P-, phosphorylated; PDLSC, periodontal ligament stem cell; shRNA/sh, short hairpin RNA; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Article Snippet: Recombinant human cynomolgus TWEAK/TNF superfamily member 12 (TNFSF12) protein (mFc tag) (cat. no. 90094-C04H; Sino Biological, Inc.) was dissolved in sterile water to obtain a stock solution concentration of 50 μ g/ml.

Techniques: Western Blot, shRNA, Two Tailed Test

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Inhibition of Fn14 and NF-κB effectively blocks TWEAK-induced alterations in PDLSC characteristics. (A) Western blot analysis was conducted to assess the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs. (B) Statistical analysis of the intensities of the protein bands shown in (A) (n=3). (C) A CCK-8 assay was performed to generate the proliferation curve of PDLSCs. (D) Statistical analysis of the OD450 values of cells from each group on day 5 of the CCK-8 assay, as presented in (C) (n=6). (G) Results of ALP staining and (E) the corresponding statistical analysis of PDLSCs after osteogenic induction (n=6). Scale bar, 400 μ m. (H) Results of Alizarin Red staining and (F) the corresponding statistical analysis of PDLSCs following osteogenic induction (n=6). Scale bar, 400 μ m. (I) Reverse transcription-quantitative PCR was used to assess the mRNA expression levels of RUNX2 , SP7 , ALP and OPG in PDLSCs, with β-actin serving as an internal reference (n=4). (J) Western blot analysis was performed to detect the protein expression levels of RUNX2, SP7, ALP and OPG in PDLSCs, and (K) the grayscale values of the gel images were semi-quantitatively analyzed, with β-actin used as an internal reference (n=3). Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. ALP, alkaline phosphatase; CCK-8, Cell Counting Kit-8; Fn14, fibroblast growth factor-inducible 14; IOD, integral optical density; NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ns, not significant; OD450, optical density at 450 nm; OPG, osteoprotegerin; P-, phosphorylated; PDLSC, periodontal ligament stem cell; RUNX2, runt-related transcription factor 2; sh, short hairpin RNA; SP7, Sp7 transcription factor; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Article Snippet: Recombinant human cynomolgus TWEAK/TNF superfamily member 12 (TNFSF12) protein (mFc tag) (cat. no. 90094-C04H; Sino Biological, Inc.) was dissolved in sterile water to obtain a stock solution concentration of 50 μ g/ml.

Techniques: Inhibition, Western Blot, CCK-8 Assay, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Cell Counting, shRNA

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Inhibition of Fn14 and NF-κB effectively blocks TWEAK-induced alterations in the microenvironmental regulatory potential of PDLSCs. (A) Expression levels of OPG (green) and RANKL (red) in PDLSCs were detected by immunofluorescence staining, followed by quantitative analysis of the MOD values for (B) RANKL and (C) OPG, and (D) the MOD ratio of RANKL/OPG (n=5). Scale bar, 200 μ m. (E) Expression levels of CD68 and CD163 in RAW264.7 macrophages were detected by immunofluorescence staining, followed by quantitative analysis of the MOD values for (F) CD68 and (G) CD163 (n=5). Scale bar, 200 μ m. Statistical analysis was performed using a one-way ANOVA. ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. Fn14, fibroblast growth factor-inducible 14; MOD, mean optical density; ns, not significant; OPGa, osteoprotegerin; PDLSC, periodontal ligament stem cell; RANKL, receptor activator of nuclear factor-κB ligand; sh, short hairpin RNA; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Article Snippet: Recombinant human cynomolgus TWEAK/TNF superfamily member 12 (TNFSF12) protein (mFc tag) (cat. no. 90094-C04H; Sino Biological, Inc.) was dissolved in sterile water to obtain a stock solution concentration of 50 μ g/ml.

Techniques: Inhibition, Expressing, Immunofluorescence, Staining, shRNA

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Inhibition of the TWEAK/Fn14/NF-κB/NLRP3 pathway enhances the functional properties of iPDLSCs. (A) Expression profile of surface markers in iPDLSCs quantified using flow cytometry. (B) Levels of TWEAK, Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3, and (C) statistical analysis of the band density values (n=3). (D) Apoptosis levels in PDLSCs, iPDLSCs, and iPDLSCs after downregulation of Fn14, NF-κB and NLRP3 were detected using the TUNEL assay, and (E) statistical analysis of the average fluorescence intensity of TUNEL was performed (n=5). Scale bar, 200 μ m. (F) A Cell Counting Kit-8 assay was used to assess the proliferative potential of PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3, and (G) statistical analysis of the OD450 values on day 5 of the experiment was performed (n=6). (H) Transwell migration assay evaluating the migratory potential of PDLSCs, iPDLSCs, and iPDLSCs after Fn14, NF-κB or NLRP3 downregulation, with (I) quantification of the number of migrated cells (n=6). Scale bar, 400 μ m. (J) Wound healing assay evaluating the migratory potential of PDLSCs, iPDLSCs, and iPDLSCs after Fn14, NF-κB or NLRP3 downregulation, with (K) quantification of the percentage of wound closure (%) (n=16). Scale bar, 1 mm. (L) ALP staining was used to evaluate the mineralization potential of PDLSCs, iPDLSCs, and iPDLSCs after downregulation of Fn14, NF-κB or NLRP3, with (M) quantification of the integral optical density of the ALP-stained images (n=6). Scale bar, 400 μ m. (N) Alizarin Red staining was used to evaluate the mineralization potential of PDLSCs, iPDLSCs, and iPDLSCs after downregulation of Fn14, NF-κB or NLRP3, with (O) quantification of the integral optical density of the Alizarin Red-stained images (n=6). Scale bar, 400 μ m. (P) Reverse transcription-quantitative PCR was used to evaluate the mRNA expression levels of RUNX2 , SP7 , ALP and OPG in PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3 (n=4). (Q) Western blotting was used to detect the expression levels of RUNX2, SP7, ALP and OPG in PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3, and (R) statistical analysis of the band density values was performed (n=3). Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. ALP, alkaline phosphatase; Fn14, fibroblast growth factor-inducible 14; IOD, integral optical density; iPDLSC, inflammatory PDLSC; NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ns, not significant; OD450, optical density at 450 nm; OPG, osteoprotegerin; P-, phosphorylated; PDLSC, periodontal ligament stem cell; RUNX2, runt-related transcription factor 2; sh, short hairpin RNA; SP7, Sp7 transcription factor; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Article Snippet: Recombinant human cynomolgus TWEAK/TNF superfamily member 12 (TNFSF12) protein (mFc tag) (cat. no. 90094-C04H; Sino Biological, Inc.) was dissolved in sterile water to obtain a stock solution concentration of 50 μ g/ml.

Techniques: Inhibition, Functional Assay, Expressing, Flow Cytometry, TUNEL Assay, Fluorescence, Cell Counting, Transwell Migration Assay, Wound Healing Assay, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, shRNA

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Effects of TWEAK and TWEAK-Fn14-IN-1 on the progression of rat periodontitis. (A) Micro-CT images of the rat maxilla, with the distance between the two red short lines representing the CEJ-ABC distance on the buccal side, and with statistical analysis of the (B) distance of CEJ-ABC (n=6), and (C) BV/TV at the root bifurcation of the maxillary second molar (n=6). Scale bar, 1 mm. Images of (D) H&E and (E) Masson's trichrome staining of rat periodontal tissues. Scale bar, 1 mm (top) or 100 μ m (bottom). (F) Representative images of TRAP/alkaline phosphatase double staining in the periodontal tissue of the rat maxillary second molar, with (G) quantification and statistical analysis of osteoclast numbers (TRAP-positive, multinucleated cells located in the bone resorption lacunae) at the mesial root (n=6). Red triangles indicate osteoclasts. Scale bar, 50 μ m. (H) Immunofluorescence staining of CD163 (green) and CD68 (red) in periodontal tissues, with (J) quantification of the mean fluorescence intensity of CD163 and (K) quantification of the mean fluorescence intensity of CD68 (n=6). Scale bar, 100 μ m. (I) Immunofluorescence staining of RUNX2 (green) and Periostin (red) in periodontal tissues, with (L) quantification of the mean fluorescence intensity of RUNX2 and (M) quantification of the mean fluorescence intensity of Periostin (n=6). Scale bar, 100 μ m. Blank represents the unmodeled group, PBS refers to the control group where PBS was used instead of TWEAK or TWEAK-Fn14-IN-1 during modeling, and TWEAK and TWEAK-Fn14-IN-1 represent experimental groups where the recombinant TWEAK protein or TWEAK-Fn14-IN-1 inhibitor was applied, respectively. Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; **** P<0.0001. Data are presented as the mean ± SD. ABC, alveolar bone crest; BV/TV, bone volume to total volume; CEJ, cementoenamel junction; Fn14, fibroblast growth factor-inducible 14; MOD, mean optical density; ns, not significant; RUNX2, runt-related transcription factor 2; TRAP, tartrate-resistant acid phosphatase; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Article Snippet: Recombinant human cynomolgus TWEAK/TNF superfamily member 12 (TNFSF12) protein (mFc tag) (cat. no. 90094-C04H; Sino Biological, Inc.) was dissolved in sterile water to obtain a stock solution concentration of 50 μ g/ml.

Techniques: Micro-CT, Staining, Double Staining, Immunofluorescence, Fluorescence, Control, Recombinant

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: Similarly, human CXCL1 (#70‐EK196), CSF3 (#70‐EK169), and CXCL8 (#70‐EK108/2) levels in cultured human keratinocyte lysates and supernatants were measured using corresponding human ELISA kits (Lianke Bio, China) according to the provided protocols.

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: 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: Similarly, human CXCL1 (#70‐EK196), CSF3 (#70‐EK169), and CXCL8 (#70‐EK108/2) levels in cultured human keratinocyte lysates and supernatants were measured using corresponding human ELISA kits (Lianke Bio, China) according to the provided protocols.

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