Fig. 2: CLSM analyses of ER-phagy induction.
a, Representative CLSM images of delivery of CNX-positive ER portions (red) to LAMP1-positive ELs (cyan) in mock-transfected MEF treated with 50 nM BafA1 and 100 nM TMR. Scale bar, 10 μm. The inset shows a 4× magnification of the merge image. b, Same as a in cells expressing FAM134BRHD-HALO (green, TMR). c, Same as b in cells expressing FAM134B-HALO. d, Same as c for FAM134BLIR-HALO. e, LysoQuant quantification of the percentage of CNX-loaded LAMP1-positive EL in a–d (mock (n = 30 cells), FAM134BRHD-HALO (n = 32 cells), FAM134B-HALO (n = 26 cells), FAM134BLIR-HALO (n = 20 cells)). N = 3 biological replicates. f–h, Same as b–d in cells expressing SEC62TMD-HALO (n = 27 cells) (f), SEC62-HALO (n = 24 cells) (g) and SEC62LIR-HALO (n = 20 cells) (h). i, LysoQuant quantification of the percentage of CNX-loaded LAMP1-positive EL in f–h. N = 3 biological replicates. Mock as in e. j–l, Same as f–i in cells expressing TEX264TMD-HALO (n = 13 cells) (j), TEX264-HALO (n = 27 cells) (k) and TEX264LIR-HALO (n = 18 cells) (l). m, LysoQuant quantification of the percentage of CNX-loaded LAMP1-positive EL in j–l. N = 3 biological replicates. Mock as in e. A one-way ANOVA with Turkey’s multiple comparisons test was performed, with F = 190.1, 193.6 and 168 for e, i and m, respectively. Adjusted P value: ****P < 0.0001, n.s., not significant (P = 0.0643, 0.3035 and 0.8759 for n.s. in e, i and m, respectively). The mean bar is shown. n, In gel native GFP fluorescence (top) and HaloTag assay in cells expressing the ER-phagy reporter HT17 (second from top) of constructs from a–l showing Halo fragment formation (high contrast) upon expression of LC3-binding-competent ER-phagy receptors FAM134B (lane 3), SEC62 (lane 6) and TEX264 (lane 9). N = 1 biological replicate. Bottom: the Coomassie loading control.
