Fig 1: Fgfr2 is not required for AT2 cell proliferation(A) Left: images using endogenous fluorescence of a representative well of AT2 cell-derived organoids from either SftpcCreERT2;R26RtdTomato (top) or SftpcCreERT2;Fgfr2fl/fl;R26RtdTomato (bottom) mice at 21 days of culture, white arrows indicate a single organoid (scale bar, 500 μm). Middle: RNA FISH for Fgfr2 on representative control (top) and Fgfr2-deficient (bottom) AT2 cell-derived organoids. Dashed white boxes mark zoomed in regions. Right: IHC for AGER and SFTPC on representative control (top) and Fgfr2-deficient (bottom) AT2 cell-derived organoids.(B) Analysis of organoid size from 11 to 21 days of culture. Each dot represents the average of organoids derived from three individual mice. Dashed black box demarcates 21 days of culture.(C and D) Analysis of organoid size and colony forming efficiency at 21 days of culture. Each dot represents the average of at least three replicate wells from one mouse for a total of n = 3 mice per group.(E) Experimental schematic outlining the timing of cytokine treatment. Transforming growth factor β (TGF-β) inhibitor was added for the first week of culture. At 7 days of culture, cytokines were added for the remainder of the experiment.(F) Left: images using endogenous fluorescence of a representative well of AT2 cell-derived organoids from either SftpcCreERT2;R26RtdTomato (top) or SftpcCreERT2;Fgfr2fl/fl;R26RtdTomato (bottom) mice at 21 days of culture after treatment with FGF7 for the last 14 days, white arrows indicate a single organoid (scale bar, 500 μm). Middle: RNA FISH for Fgfr2 on representative control (top) and Fgfr2-deficient (bottom) AT2 cell-derived organoids. Dashed white boxes mark zoomed in regions. Right: IHC for AGER and SFTPC on representative control (top) and Fgfr2-deficient (bottom) AT2 cell-derived organoids.(G) Analysis of organoid size from 11 to 21 days of culture. Each dot on this graph represents the average from three individual mice, n = 3. Dashed black box demarcates 14 days of culture.(H and I) Analysis of organoid size in untreated versus FGF7 treated control and Fgfr2-deficient (KO) organoids at 14 days of culture highlighted in (G). Each dot represents the average of at least three replicate wells from one mouse for a total of n = 3 mice per group.(J) Representative images of AT2 cell-derived organoids from either SftpcCreERT2;R26RtdTomato (top) or SftpcCreERT2;Fgfr2fl/fl;R26RtdTomato (bottom) mice at 21 days of culture after treatment with IL-1α, IL-1β, or TNF-α for the last 14 days of culture (scale bar, 500 μm).(K) Organoid size at 21 days of culture. Each dot represents the average of at least three replicate wells from one mouse for a total of n = 3 mice per group.All quantification data are represented as mean ± SEM. Two-tailed t tests: ns, not significant; *p ≤ 0.05, **p ≤ 0.01. Scale bars, 50 μm unless otherwise noted.
Fig 2: FGF7 promoted muscle regeneration after injury in mice. (A) Experimental schematic. The TA muscles of 8‐week‐old mice were infected with CTX at 0 days and FGF7 recombinant protein (control group was infected with 0.9% NaCl) at 1 day, and samples were collected at 0, 3, 5 and 15 days. EdU was injected intraperitoneally for two consecutive days before analysis. (B) H&E staining of TA muscle at 0, 3, 5 and 15 days after injury (scale bar: 50 μm) (n = 5). (C) Immunofluorescence staining with EdU (green), Pax7 (red) and DAPI (blue) of TA muscle at 3 days after injury, and the ratio of Pax7+ cells and EdU+ versus Pax7+ cells was quantified. The scale bar is 20 μm (n = 4). (D) Immunofluorescence staining with eMyHC (red), laminin (green) and DAPI (blue) of TA muscle at 5 days after injury, and the average cross‐sectional area (CSA) and ratio of eMyHC+ myofibre (vs. laminin+ myofibres) were quantified. The scale bar is 25 μm (n = 4). (E) Immunofluorescence staining with laminin (green) and DAPI (blue) of TA muscle at 15 days after injury, and the myofibre CSA was measured with ImageJ. The scale bar is 50 μm (n = 3) (*P < 0.05 and ** P < 0.01). Data are presented as mean ± SD.
Fig 3: FGF7 mediated the interaction between FAPs and myogenic cells. (A) UMAP plots of FGF7 and FGF2 expression in porcine muscle mononuclear cells. (B) Dot plot of FGF members in human muscle scRNA‐seq data published by Rubenstein et al. 14 The dot size was proportional to the per cent of FGF expression in the corresponding cell group. And the colour showed the average expression of FGFs. (C) Dot plot of FGF members in mice skeletal muscle scRNA‐seq data, as well as the violin plot of FGF7 expression in mice FAPs collected at 0, 2, 5 and 7 days after injury published by De Micheli et al. 15 (D) Violin plot of FGF7 between LD and SOL mononuclear cells. (E) Western blot analyses of FGF7 in LD and SOL muscles in 3‐day‐old piglets and EDL and SOL muscles in 8‐month‐old mice. The quantification of FGF7 protein levels was normalized to both β‐tubulin and the first sample on the left. (F) Immunofluorescence staining of FGFR2 in LD‐MuSCs and SOL‐MuSCs, and the ratio of FGFR2+ cells from LD‐MuSCs and SOL‐MuSCs were quantified. The scale bar is 50 μm (n = 3). Data are presented as mean ± SD (*P < 0.05).
Fig 4: FGF7 delayed d‐gal‐induced muscle ageing in mice. (A) Experimental schematic. The 8‐week‐old mice were treated with continuous intraperitoneal (i.p.) injection of d‐gal for 6 weeks. During the last 2 weeks, twice‐weekly TA intramuscular (i.m.) injections of FGF7 were performed. Mice running experiments were performed after three injections of FGF7. And samples for H&E staining were collected 1 day after the fourth injection of FGF7. Following successful d‐gal‐induced ageing, TA was injected with CTX. FGF7 injection was performed 1 day later, and EdU was injected intraperitoneally for two consecutive days before analysis. Samples for H&E staining and immunofluorescence staining were collected 3 days after CTX injection. (B) Body weight changes among 6 weeks of d‐gal i.p. injection (n = 6). (C) H&E staining of TA muscle after four injections of FGF7, and the myofibre CSA was analysed with ImageJ. The scale bar is 100 μm (n = 5). (D) Distance running to exhaustion (m) and time running to exhaustion (min) (n = 6). (E) H&E staining of TA muscle at 3 days after injury (scale bar: 20 μm) (n = 5). (F) Immunofluorescence staining with EdU (green), Pax7 (red) and DAPI (blue) of TA muscle at 3 days after injury, and the ratio of Pax7+ cells and EdU+ versus Pax7+ cells was quantified. The scale bar is 20 μm (n = 5) (*P < 0.05, ** P < 0.01 and *** P ≤ 0.001). Data are presented as mean ± SD.
Fig 5: FGF7–FGFR2 promoted proliferation of myogenic cells. (A) Immunofluorescence staining with EdU (red) and Hoechst (blue), Ki67 (red) and DAPI (blue) in porcine MuSCs treated with a series of concentrations of FGF7 recombinant protein (0, 1, 10 and 100 ng/mL). The scale bar is 100 μm. (B) Quantification of the proportion of Ki67+ cells in porcine MuSCs treated with a series of concentrations of FGF7 recombinant protein (0, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ng/mL) (n = 3). (C) Western bolt analysis of Ki67 in porcine MuSCs treated with FGF7 recombinant protein (0, 1, 10 and 100 ng/mL). (D) Quantification of the proportion of EdU+ cells in porcine MuSCs treated with FGF7 recombinant protein (0, 1, 10 and 100 ng/mL) (n = 3). (E) Immunofluorescence staining with Ki67 (red) and DAPI (blue) in porcine MuSCs treated with si‐pFGFR2 and 10‐ng/mL FGF7 recombinant protein, and the mRNA level of porcine FGFR2 was detected to show its knockdown efficiency, and the ratio of Ki67+ cells was quantified. The scale bar is 200 μm (n = 3). (F) Immunofluorescence staining with EdU (red) and Hoechst (blue) in C2C12 cell line treated with si‐mFGFR2 and 10‐ng/mL FGF7 recombinant protein, and the mRNA level of mice FGFR2 was detected to show its knockdown efficiency, and the ratio of EdU+ cells was quantified. The scale bar is 100 μm (n = 3) (*P < 0.05 and ** P < 0.01). Data are presented as mean ± SD.
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