Autophagy induced foci formation by HaloTagged autophagy proteins. (A) Live-cell imaging of GFP-LC3 and JF646-labeled HaloTagged proteins in control (Ctr) and upon treatment with rapamycin (Rapa, 100 nM for 2 h) or rapamycin + bafilomycin (Rapa+Baf, 100 nM each, for 2 h). Both treatments show an expected increase in autophagy and LC3 foci. Scale bar = 10 μm. (B) IF with anti-LC3B antibody and HaloTag JF646 labeling for Halo-ATG5, Halo-ATG16, and Halo-WIPI2 cell lines in control (Ctr) and upon treatment with rapamycin (Rapa, 100 nM for 2 h) or rapamycin + bafilomycin (Rapa+Baf, 100 nM each, for 2 h). Both treatments show an expected increase in autophagy and LC3 foci. Scale bar = 10 μm. (C) Representative images of JF646-labeled HaloTagged proteins under control (Ctr) or EBSS starvation media, demonstrating foci-forming ability upon autophagy induction. Scale bar = 10 μm. (D) Quantification of autophagy factor foci from live-cell imaging (A). Data represent the mean ± 1SD of three biological replicates. (E) Quantification of LC3 foci from live-cell imaging (A; N = 3, mean ± SD). (F) Quantification of autophagy factor foci from live-cell imaging (C; N = 3, mean ± SD).
Figure S2.

Autophagy induced foci formation by HaloTagged autophagy proteins. (A) Live-cell imaging of GFP-LC3 and JF646-labeled HaloTagged proteins in control (Ctr) and upon treatment with rapamycin (Rapa, 100 nM for 2 h) or rapamycin + bafilomycin (Rapa+Baf, 100 nM each, for 2 h). Both treatments show an expected increase in autophagy and LC3 foci. Scale bar = 10 μm. (B) IF with anti-LC3B antibody and HaloTag JF646 labeling for Halo-ATG5, Halo-ATG16, and Halo-WIPI2 cell lines in control (Ctr) and upon treatment with rapamycin (Rapa, 100 nM for 2 h) or rapamycin + bafilomycin (Rapa+Baf, 100 nM each, for 2 h). Both treatments show an expected increase in autophagy and LC3 foci. Scale bar = 10 μm. (C) Representative images of JF646-labeled HaloTagged proteins under control (Ctr) or EBSS starvation media, demonstrating foci-forming ability upon autophagy induction. Scale bar = 10 μm. (D) Quantification of autophagy factor foci from live-cell imaging (A). Data represent the mean ± 1SD of three biological replicates. (E) Quantification of LC3 foci from live-cell imaging (A; N = 3, mean ± SD). (F) Quantification of autophagy factor foci from live-cell imaging (C; N = 3, mean ± SD).

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