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. 2018 Jan 22;44(2):217–232.e11. doi: 10.1016/j.devcel.2017.11.024

Figure 4.

Figure 4

CCPG1 Is Recruited into Autophagosomes from the ER

(A) A549 cells were transfected with siCtrl or siCCPG1 and, at 24 hr post-transfection, either left untreated or starved for 1 hr in EBSS, then stained for endogenous CCPG1. Cells with CCPG1 foci were scored (n = 3, ± SEM, ∗p < 0.05, two-tailed paired sample t tests). Scale bar, 20 μm.

(B) A549 or A549 GFP-DFCP1 cells were starved for 1 hr in EBSS and co-stained for endogenous CCPG1 and, for A549 cells, the indicated marker, then imaged by confocal microscopy. Arrowheads indicate co-localizing foci. Scale bars, 10 μm.

(C) HeLa GFP-CCPG1 cells (wild-type [WT]) or indicated ATG8 (mtLIR) or FIP200 (mtFIR1+2) binding-deficient variants were starved, stained with ER tracker, and imaged by confocal microscopy. Automated quantification of GFP foci per cell was performed as described in the STAR Methods (n = 3, ± SEM, ∗∗∗p < 0.001, one-way ANOVA with Tukey's post-hoc test). Scale bar, 20 μm.

(D) HeLa GFP-CCPG1 mCherry-ER cells were starved, stained for LC3B, and then imaged by 3D-SIM. Top left panel shows a reconstructed region of cell observed from above. The rightmost panels are zoomed images of the white boxed region. Lower panels show a cross-section along the white dashed line. Scale bars, 5 μm and 0.5 μm (zoomed).

(E) HeLa GFP or GFP-CCPG1 cells, WT or indicated mutants, introduced in (D), were starved for 3 hr and blotted for GFP (left). Blots were quantified by densitometry for GFP:tubulin ratios (right) (n = 3, ± SEM, ∗p < 0.05, #not significant, two-tailed t test).

(F) HeLa GFP or GFP-CCPG1 cells (WT or indicated mutants) were starved for 1 hr, co-stained for LC3B, and imaged by confocal microscopy. Pearson's coefficient for colocalization of GFP foci with LC3B was derived as described in the STAR Methods. White dashed lines indicate the outline of GFP-positive cells (n = 3, ± SEM, ∗∗∗p < 0.001, one-way ANOVA with Tukey's post-hoc test). Scale bar, 20 μm.

(G) A549 cells were left untreated or starved for 4 hr in EBSS with or without 0.1 μM bafilomycin A1 (BafA1) and immunoblotted.

(H) WT or ΔATG5 A549 clones were left untreated or starved for 4 hr in EBSS and immunoblotted.

(I) A549 cells were transfected with siRNA for 48 hr and then starved and immunoblotted as shown (I and II indicate unlipidated and lipidated forms of LC3B, respectively). See also Figure S3.