Fig. 2: ASB2α-mediated degradation of FLNa and FLNb confers specific morphological features to Th2 lymphocytes.

a–d Th1, Th2, Th17, and Treg ctrl lymphocytes were seeded onto VCAM-1 coated 384-well plates and analyzed by immunofluorescence with antibodies to FLNa and FLNb or phalloidin. Representative fluorescent images of one experiment out of 3 (a), FLNa MFI and FLNb MFI (b), radar plot with FLNa MFI, FLNb MFI, cell area, cell perimeter, and width to length ratio (relative to the values measured in Th2 lymphocytes) (c) and violin plots of cell area, cell perimeter and width to length ratio (d) are shown. e–g Ctrl and ASB2 cKO Th2 lymphocytes were allowed to adhere in 384-well plates coated with vitronectin, fixed, and stained for FLNa, FLNb, and F-actin. Representative fluorescent images of one experiment out of 4 (e), radar plot with FLNa MFI, FLNb MFI, cell area, cell perimeter, and width-to-length ratio (relative to the values measured in ctrl Th2 lymphocytes) (f), and violin plots with FLNa MFI, FLNb MFI, cell area, cell perimeter and width to length ratio (g) are shown. h, i Human naive CD4 + T lymphocytes and in vitro generated Th2 lymphocytes were seeded onto VCAM-1 coated 384-well plates and analyzed by immunofluorescence with antibodies to FLNa or phalloidin. Violin plots of FLNa MFI (h), cell area, cell perimeter, and width-to-length ratio (i) are shown. Scale bar, 20 µm. Data are mean ± SEM. Sample size: Th1 = 2097, Th2 = 3397, Th17 = 1284 and Treg = 1918 for (b) (left); Th1 = 222, Th2 = 660, Th17 = 750 and Treg = 951 for (b) (right); Th1 = 1002, Th2 = 2405, Th17 = 486 and Treg = 560 for (d) (left); ctrl = 7189 and cKO = 6878 for (f) (FLNa MFI); ctrl = 5159 and cKO = 3139 for (f) (FLNb MFI); ctrl = 2589 and cKO = 1605 for (f) (width/length ratio, area, perimeter); naive = 1141 and Th2 = 1137 for i. p values were calculated using the two-sided Mann–Whitney t-test except in (g, j) where the Wilcoxon matched-pairs signed rank test was used. Source data are provided as a Source Data file.