Figure 2
From: Synergy between B cell receptor/antigen uptake and MHCII peptide editing relies on HLA-DO tuning
DO tunes DMfree/CLIPfreq in LAMP1+ compartments despite surface display of CLIP. (a) Representative 3D-SIM overlay views of fixed/permeabilized T2DR4DMDO cells co-stained for LAMP1 and CLIP. Cells analyzed (Ncell): 18 CLIPhi and 19 CLIPlo cells in 10 reconstructed 3D images. Experimental replicates (n) = 3. (b) Representative 3D-SIM overlay views of fixed/permeabilized 1C3 or 2D7 cells co-stained for LAMP1, DM and DO (see also Supplementary Fig. 2). n = 3. (c) Comparison of %DO co-localized with the indicated endosomal marker in 1C3 and 2D7 cells. Ncell (from left to right): 13, 8, 16, 15, 14, 16. (d) Comparison of %DO co-localized with DM in 1C3 and 2D7 cells. Ncell: 25, 21. (e) Flow cytometric analysis (n = 4) of fixed/permeabilized 1C3, 2D7, or T2DR4DM cells co-stained for DM and DO. The ratio of mean FIDO to mean FIDM for each cell line was normalized to that for 2D7 cells. (f) Comparison of %DM co-localized with the indicated marker in 1C3 and 2D7 cells. Ncell: 13, 8, 4, 12, 18, 21, 14, 18, 14. (g) Comparison %DM co-localized with DO in 1C3 and 2D7 cells. Ncell: 25, 21. (h) 3D-SIM analysis (n = 3) of fixed/permeabilized 1C3 or 2D7 co-stained for LAMP1 and CLIP. Shown are overlay, single channel, and rotated zoom-in views. (i) Flow cytometric analysis (n = 3) of CLIPsrf (line) and CLIPtot (filled) in T2, 2D7 or T2DR4 cells. MFI of CLIPint = MFI of CLIPtot – MFI of CLIPsrf. (j,k) Comparisons of %CLIP or %DR co-localized with the indicated marker in 2D7 and T2DR4 cells (see also Supplementary Fig. 3). Ncell (same for j,k): 10, 11, 4, 7, 30, 37. (l) The functional correlation between varied levels of DM/DO and their regulation of CLIP removal. Transparency is related to protein levels per cell. Data are represented as mean ± SEM. ns: non-significant, p > 0.05; *p < 0.05, ****p < 0.0001.
