Figure 6

Rescue of GPI-anchored protein expression and TLR4 signaling by PIGH re-expression. (a) Western blot analysis of GPI-negative (GPI-) BLaER1 clones that were either transduced with a lentivirus encoding for GFP, PIGH-Flag, or PIGA-Flag, or not transduced (/) as a negative control. One representative blot of three is shown. Full-length blots are presented in Supplementary Figure S9. (b) GPI-negative BLaER1 cells stably expressing GFP, PIGH-Flag, or PIGA-Flag were analyzed by flow cytometry. The percentage of CD48-expressing cells among GFP-positive cells is shown as the mean + SD from n = 3 independent measurements. For comparison, CD48 surface expression of a GPI-positive (GPI +) BLaER1 monoclonal cell line is shown on the right-hand side. Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to determine statistical significance. Results from comparing cells transduced with either GFP, PIGH-Flag, or PIGA-Flag with the negative control (not transduced, /) are shown (ns – not significant, ****p ≤ 0.0001). (c) GPI-negative BLaER1 cells stably expressing GFP or PIGH-Flag were transdifferentiated for 5 days and then analyzed by flow cytometry. The percentage of CD14-expressing cells among GFP-positive cells is shown as the mean + SD from n = 3 independent measurements. (d) IL-6 and IL-1β in the supernatant were determined by ELISA 16 h after the stimulation of BLaER1 monoclonal cell lines, stably expressing GFP or PIGH-Flag, with LPS or medium control (0). Values correspond to the mean + SD of n = 3 independent experiments. (e–g) BLaER1 clones stably expressing GFP or PIGH-Flag were stimulated for 16 h with four different TLR7/TLR8 ligands and the supernatant was used to determine IL-6 (e) and IL-1β (g) concentrations by ELISA, or type I IFN concentration by HEK blue assay (f). Data for stimulation with R848 40 ng/ml and 200 ng/ml, TL8-506 20 ng/ml, and CL264 200 ng/ml are depicted as mean + SD of n = 2 independent experiments. For all other stimuli and concentrations, the mean + SD of n = 3 independent experiments is shown. (c–g) BLaER1 cells stably expressing PIGH-Flag were compared to GFP-expressing control cells using unpaired t tests. Statistical significance is depicted as follows: ns—not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.