Fig. 6.

Atg8 proteins differentially regulate Fn14. a HeLa cells were transfected with siNT or siRNA against individual Atg8 isoforms as indicated, immunostained for Fn14, and analyzed by confocal microscopy (scale bar, 20 µm). b HeLa cells transfected with siRNA against GABARAP (siGB) were treated with TWK and BafA as indicated, immunostained for Fn14 and ERGIC-53, and analyzed by confocal microscopy (scale bar, 20 µm) (upper panel). Large magnification of stained cells is presented in the right column. ERGIC-localized Fn14 fluorescence was quantified from at least 10 fields (lower panel) (**P < 0.0025; ***P < 0.0001, comparisons by ANOVA. siNT—control, n = 9 cells; BafA, n = 14 cells; TWK, n = 19 cells; TWK baf, n = 22 cells. SiGB—control, n = 23 cells; BafA, n = 27 cells; TWK, n = 32 cells; TWK + BafA, n = 28 cells; mean ± s.e.m.). c HeLa cells transfected with siRNA against GATE-16 (siGATE-16) were treated with TWK as indicated, immunostained for Fn14 and TRAF2, and analyzed by confocal microscopy (scale bar, 20 µm). d HeLa cells transfected with siNT, siGATE-16, or siGB were immunostained for LC3B and Fn14, and analyzed by 3D STORM (scale bar, 300 nm). e, f Cells were transfected as with siNT or siGATE-16 (e) or siGB (f) and after 24 h were transfected with luciferase expression constructs as detailed under Methods, treated with TWK, and further subjected to luciferase assay as detailed to assess NF-kB activity (*P < 0.01, comparisons by T test; mean ± s.e.m.; n = 3 biological repeats and n = 2 biological repeats performed in triplicates, respectively)