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) Volcano plots showing KLF4 affinity purification-mass spectrometry (KLF4 AP-MS) data from serum-starved HaCaT keratinocytes treated with FGF7 for 6 h or vehicle vs . the respective IgG control. Enriched prey proteins are located in the upper right quadrant, with KLF4 highlighted (N = 3, one-sided Student’s t -test, FDR<0-05). B) Volcano plot depicting FGF7-dependent changes in KLF4-associated proteins (KLF4 AP-MS data) under the same experimental conditions. p < 0.05 (two-sided Student’s t -test), filtering threshold ≥3 unique tryptic peptides per protein. C) Proteins, which also showed a significantly different abundance in the KLF4 interactome upon FGF7 treatment in an independent experiment. D) Schematic representation of the luciferase reporter vector harboring 4 KLF4 binding sites in the promoter. E) Luciferase activity in lysates of serum-starved HaCaT keratinocytes, stably transduced with lentiviruses including a luciferase reporter gene preceded by KLF4 response elements. Cells were treated for 8, 16 or 24 h with FGF7 or vehicle (N = 6). F) Luciferase activity in lysates of HaCaT keratinocytes, stably transduced with lentiviruses containing a luciferase reporter gene preceded by KLF4 response elements. Cells had been serum-starved and pre-treated for 2 h with the MEK1/2 inhibitor U0126 or vehicle, followed by a 6 h treatment with FGF7 or vehicle (N = 6). G) IL6 promoter cloning strategy showing the deletion of two KLF4 binding sites (BS) and the insertion of the IL6 promoter fragment into the firefly luciferase lentiviral vector. H) Luciferase activity in lysates of HaCaT keratinocytes, stably transduced with lentiviruses containing the IL6 promoter fragment with or without KLF4 BS in front of a luciferase gene. Cells were serum-starved and treated for 6 h with FGF7 or vehicle (N = 6). I) Luciferase activity in lysates of serum-starved HaCaT keratinocytes, stably transduced with lentiviruses containing the IL6 promoter fragment with or without KLF4 BS in front of a 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 SD. Non-significant (ns), *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001 (Mann-Whitney U test (D, G; normalized to respective control), or 2-way ANOVA with Bonferroni’s multiple comparisons test (E; H).

Article Snippet: The beads were resuspended in 80 μl binding buffer, to which 9.21 μg of KLF4 antibody (Cell Signaling) or 9.21 μg of rabbit control IgG antibody (Merck, Darmstadt, Germany) was added.

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Control, Luciferase, Plasmid Preparation, Binding Assay, Activity Assay, Stable Transfection, Transduction, Cloning, Incubation, MANN-WHITNEY

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) ChIP-seq data from the Gene Transcription Regulation Database (GTRD) showing the number of KLF4 binding sites (BS) in the promoter regions (−100 to +10 bp relative to the TSS) of FGF7-suppressed genes. B) RT-qPCR for IL6, RSAD2, ISG20 relative to RPL27 using RNA from serum-starved HaCaT keratinocytes or HPKs, which had been transfected with scrambled (scr) or KLF4 siRNA (N = 6; HPKs from two donors). C) Luciferase activity in lysates of HaCaT keratinocytes, stably transduced with lentiviruses containing the IL6 promoter fragment with or without KLF4 binding sites in front of a luciferase gene. Cells had been transfected with scrambled (scr) or KLF4 siRNA, serum-starved, and treated for 6 h with FGF7 or vehicle (N = 6). Data information: Graphs show mean and SD. Non-significant (ns), **P < 0.01, ****P < 0.0001 (Mann-Whitney U test (B, C)).

Article Snippet: The beads were resuspended in 80 μl binding buffer, to which 9.21 μg of KLF4 antibody (Cell Signaling) or 9.21 μg of rabbit control IgG antibody (Merck, Darmstadt, Germany) was added.

Techniques: ChIP-sequencing, Binding Assay, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Stable Transfection, Transduction, MANN-WHITNEY

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) Western blot of lysates from serum-starved HaCaT keratinocytes, transfected with scrambled (scr) or KLF4 siRNA mix and treated at 48 h post transfection with FGF7 or vehicle for 15 min. Graphs show densitometric quantification of KLF4, FGFR2 and total FRS2α band intensities normalized to the intensity of α-tubulin (upper panel) or p-FRS2α/FRS2α and p-ERK1/2/total ERK1/2 ratios (lower panels) (N = 3). B) RT-qPCR for DUSP6 and INHBA relative to RPL27 using RNA from serum-starved HaCaT keratinocytes, transfected with scr or KLF4 siRNA and treated at 48 h post transfection with FGF7 or vehicle for 6 h (N = 3). C, D) RT-qPCR for FGFR2 and FRS2A using RNA from serum-starved HaCaT keratinocytes or HPKs, transfected with scr or KLF4 siRNA (N = 3-6; HPKs from two donors). E) ChIP-seq data from the GTRD showing the number of KLF4 binding sites (BS) in the promoter regions (−500/−100 to +10/+50 bp relative to the TSS) of the FRS2A and FGFR1 - FGFR4 genes. F) RT-qPCR for FGFR2 and FRS2A using RNA from serum-starved HaCaT keratinocytes, incubated with membrane-permeable KLF4-TAT, FITC-TAT, or vehicle for 8 h (N = 6). Data information: Graphs show mean and standard deviation (SD). Non-significant (ns), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t-test (A, normalized to respective control; C, D), one-way ANOVA with Bonferroni’s multiple comparisons test (F) or 2-way ANOVA with Bonferroni’s multiple comparisons test (B)).

Article Snippet: The beads were resuspended in 80 μl binding buffer, to which 9.21 μg of KLF4 antibody (Cell Signaling) or 9.21 μg of rabbit control IgG antibody (Merck, Darmstadt, Germany) was added.

Techniques: Western Blot, Transfection, Quantitative RT-PCR, ChIP-sequencing, Binding Assay, Incubation, Membrane, Standard Deviation, Control

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) Scheme depicting the FGF7–FGFR2b-ERK1/2-KLF4 signaling axis and its effect on inflammatory gene expression keratinocytes. The icons were obtained from BioRender. Werner, S. (2026) https://BioRender.com/kreppyv . B) Representative immunofluorescence stainings of normal human skin for FGFR2 (purple) and KLF4 (green). Scale bar: 20 μm.

Article Snippet: The beads were resuspended in 80 μl binding buffer, to which 9.21 μg of KLF4 antibody (Cell Signaling) or 9.21 μg of rabbit control IgG antibody (Merck, Darmstadt, Germany) was added.

Techniques: Gene Expression, Immunofluorescence