Fig. 4: CLEC-2/PDPN signaling controls intrinsic FRC mechanics for morphological adaption.
From: Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics
a, Optical tweezers generate membrane tethers of FRCs; scale bar (10 µm, top panels). Stationery (bottom left) and tether (bottom right) bead displacement (red circles). Scale bar, 2 µm (bottom panels). b, Trap force (pN µm−1) of FRCs pretreated with CLEC-2. Box plot indicates median, interquartile range, and minimum/maximum. Mann–Whitney test (two tailed), ****P = 1.00 × 10−6. n indicates cell for CLEC-2− (n = 35) and CLEC-2+ (n = 69) over five independent experiments. c, Trap force (pN µm−1) of PDPN short hairpin RNA (shRNA) knockdown (KD) FRC. Box plot indicates median, interquartile range, and minimum/maximum. Mann–Whitney test (two tailed), ****P = 1.00 × 10−6. n indicates cell for control (n = 86), PDPN KD (n = 64) over five independent experiments. d, Caveolae structures in FRCs for DAPI (blue), F-actin (red), Caveolin-1 (magenta), and EHD2 (green). Representative images of two independent experiments. Scale bars, 20 µm (zoom-ins (white box), 10 µm). e,f, Swelling of PDPN CTRL FRCs with or without pretreatment CLEC-2 (e) and PDPN shRNA KD FRCs (f). Initial size (left, white circle) after swelling (black circle). Scale bars, 25 µm. Change in diameter ratio (middle, mean ± s.e.m.). Diameter ratio (right) of control and CLEC-2-treated cells (e) or PDPN KD cells (f) at 15 min after swelling (orange dotted line). Box plot indicates median, interquartile range, and minimum/maximum. e, One-way ANOVA with Tukey’s multiple comparisons, *P = 0.0277. n indicates cell for CLEC-2− isotonic control (ISO) (n = 48), CLEC-2− hypotonic solution (HYPO) (n = 56), CLEC-2+ ISO (n = 55), and CLEC-2+ HYPO (n = 42) cells analyzed over five independent experiments. f, One-way ANOVA with Tukey’s multiple comparisons, *P = 0.0212. n indicates cell for control ISO (n = 19), control HYPO (n = 47), PDPN KD ISO (n = 19), and PDPN KD HYPO (n = 54) cells analyzed over five independent experiments. g, Individually labeled FRCs in vivo. Second harmonic (ECM, magenta), CFP (cyan), YFP (yellow), GFP (green). Representative images of three LNs over two independent experiments. Scale bars, 10 µm (zoomed-in region (white box), 5 µm).
