Journal: bioRxiv
Article Title: An FGF7-FGFR2-KLF4 feedback loop sustains anti-inflammatory signaling in epithelial cells
doi: 10.64898/2026.03.20.711763
Figure Lengend Snippet: A) RT-qPCR for IL6 relative to RPL27 using RNA from serum-starved HaCaT keratinocytes, which had been pre-treated for 1 h with actinomycin D or vehicle and incubated for 6 h with FGF7 or vehicle (N = 12 per treatment group). B) RT-qPCR for IL6 using RNA from serum-starved HaCaT keratinocytes, pre-treated for 2 h with LY294002 (PI3K inhibitor), U0126 (MEK1/2 inhibitor) or vehicle, followed by 6 h FGF7 treatment (N = 6). C) RT-qPCR for IL6 using RNA from HaCaT keratinocytes, treated for 6 h with the FGFR kinase inhibitors BGJ398 (N = 9), AZD4547 (N = 3) or erdafitinib (N = 3) in 10% FBS. D) RT-qPCR for IL6 using RNA from serum-starved WT and FGFR2 KO HaCaT cell lines, treated for 6 h with FGF7 or vehicle (N = 9; 3 different WT and KO cell lines). E) RT-qPCR for IL6 using RNA from serum-starved HaCaT or human primary keratinocytes (HPKs), pre-treated for 3 h with FGF7 or vehicle and incubated for 3 h with poly(I:C) or vehicle (N = 6; HPKs from two donors). F) RT-qPCR for IL6 using RNA from serum-starved HaCaT keratinocytes, pre-treated for 3 h with FGF7 or vehicle and incubated for 3, 6 or 12 h with TNFα or vehicle (N = 3). G) Relative IL-6 levels (ELISA) in conditioned medium from serum-starved HaCaT keratinocytes, pre-treated for 3 h with FGF7 or vehicle and incubated for 6 or 12 h with TNFα or vehicle (N = 3). H) IL6 promoter cloning strategy showing 2044 or 1057 base pair (bp) fragments including the transcription start site (TSS), inserted into a firefly luciferase vector. I) Luciferase activity in lysates of HaCaT keratinocytes, stably transduced with lentiviruses containing the long or short IL6 promoter fragment in front of the luciferase gene. Cells were serum-starved and treated for 6 h with FGF7 or vehicle (N = 6). J, K) Luciferase activity in lysates of HaCaT keratinocytes, stably transduced with lentiviruses containing the long or short IL6 promoter fragment in front of the luciferase gene. Cells had been serum-starved and pre-treated for 3 h with FGF7 or vehicle and incubated for 6 h with poly(I:C), TNFα, or vehicle (N = 6). Data information: Graphs show mean and standard deviation (SD). ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Mann-Whitney U test (A, B, I, normalized to respective control), one-way ANOVA with Bonferroni’s multiple comparisons test (C), 2-way ANOVA with Bonferroni’s multiple comparisons test (D; E, normalized to respective control; F; J; K), or Student’s t-test (G, normalized to respective control)).
Article Snippet: Cells were treated with human FGF7 or FGF10 (10 ng/ml, both from Peprotech, Rocky Hill, NJ), poly(I:C) (1 μg/ml, InvivoGen, San Diego, CA), TNFα (20 ng/ml, Peprotech), 2’3’-cGAMP (10 μg/ml, InvivoGen), 3p-hpRNA (1 μg/ml, InvivoGen), actinomycin D (1 μg/ml; Sigma-Aldrich, St. Louis, MO), the FGFR kinase inhibitors BGJ398 (3.6 μM), AZD4547 (10 μM) or erdafitinib (5 μM) (all from Selleckchem, Frankfurt, Germany), the MEK1/2 inhibitor U0126 (10 μM; Calbiochem), the PI3K inhibitor LY294002 (5 μM; Calbiochem), FITC-LC-TAT (100 nM, AnaSpec, Fremont, CA) and/or KLF4-TAT (100 nM, Peprotech).
Techniques: Quantitative RT-PCR, Incubation, Enzyme-linked Immunosorbent Assay, Cloning, Luciferase, Plasmid Preparation, Activity Assay, Stable Transfection, Transduction, Standard Deviation, MANN-WHITNEY, Control
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