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. 2021 Sep 1;10:e71642. doi: 10.7554/eLife.71642

Figure 4. Disrupting autophagy leads to the accumulation of Akita puncta that colocalize with SEC24C, and LC3B, but not SEC24A.

(A) U2OS cells expressing Akita-sfGFP and mCherry-SEC24C or mCherry-SEC24A were treated with MRT68921 for 3.5 hr. Arrowheads in the inset indicate Akita puncta colocalizing with SEC24C. (B) Bar graph showing the % of cells with multiple Akita-sfGFP puncta colocalizing with mCherry-SEC24C or mCherry-SEC24A puncta 0 or 3.5 hr after treatment with MRT68921. (C) Cells expressing Akita-sfGFP and mCherry-SEC24C were depleted of RTN3 by RNAi and treated with MRT68921 for 0 or 3.5 hr. The % of cells showing multiple Akita-sfGFP puncta colocalizing with mCherry-SEC24C puncta was quantified at the indicated time points. (D) Cells expressing Akita-sfGFP and mCherry-LC3B were depleted of RTN3 and treated with MRT68921 for 0 or 3.5 hr. A representative image for control cells (3.5 hr) is shown. Arrowhead in the inset shows an Akita puncta colocalizing with LC3B. (E) Bar graph showing the % of cells with multiple Akita-sfGFP puncta colocalizing with mCherry-LC3B puncta for the data shown in (D). Scale bars in (A) and (D), 10 µm. Error bars in (B), (C), and (E) represent SEM; n = 3 independent experiments. Approximately 20–30 cells/experiment were examined. NS: not significant (p≥0.05); *p<0.05, **p<0.01, Student’s unpaired t-test.

Figure 4.

Figure 4—figure supplement 1. Akita also colocalizes with SEC23A.

Figure 4—figure supplement 1.

(A) Representative confocal images for the data at 0 hr in Figure 4B. Arrowheads in the inset indicate Akita puncta colocalizing with SEC24C. (B) Bar graph showing the % of Akita-sfGFP puncta colocalizing with mCherry-SEC24C or mCherry-SEC24A puncta for the data in Figure 4B at 0 hr. (C) Akita colocalized with SEC24C and SEC23A. Cells expressing Akita-sfGFP and mCherry-SEC24C or mRuby-SEC23A were analyzed by confocal microscopy. Arrowheads in the inset indicate Akita puncta colocalizing with SEC24C (top) or SEC23A (bottom). (D) Bar graph showing the % of Akita-sfGFP puncta colocalizing with mCherry-SEC24C or mRuby-SEC23A puncta for the data shown in (C). Scale bars in (A) and (C), 10 µm. Error bars in (B) and (D) represent SEM; n = 3 independent experiments. Approximately 20–30 cells/experiment were analyzed. NS: not significant (p≥0.05); *p<0.05, Student’s unpaired t-test.
Figure 4—figure supplement 2. Proinsulin colocalizes with both SEC24C and SEC24A.

Figure 4—figure supplement 2.

(A) Cells expressing proinsulin-sfGFP and mCherry-SEC24C or mCherry-SEC24A were examined by confocal microscopy. Arrowheads indicate proinsulin puncta colocalizing with SEC24C or SEC24A. (B) Bar graph showing the % of proinsulin-sfGFP puncta colocalizing with mCherry-SEC24C or mCherry-SEC24A puncta for the data shown in (A). (C) Bar graph showing the % of proinsulin-sfGFP puncta of different sizes that colocalize with mCherry-SEC24A puncta for the data shown in (A). Scale bar in (A), 10 µm. Error bars in (B) and (C) represent SEM; n = 3 independent experiments. Approximately 20–30 cells/experiment were examined. NS: not significant (p≥0.05); **p<0.01, Student’s unpaired t-test.
Figure 4—figure supplement 3. Akita fails to colocalize with SEC24C, and LC3B, in the absence of RTN3.

Figure 4—figure supplement 3.

(A) Cells expressing Akita-sfGFP and mCherry-SEC24C were depleted of RTN3 by siRNA and treated with MRT68921 for 3.5 hr. A representative confocal image is shown. Quantitation is shown in Figure 4C. (B) Cells expressing Akita-sfGFP and mCherry-LC3B were depleted of RTN3 by siRNA and treated with MRT68921 for 3.5 hr. The quantitation of data is shown in Figure 4E. (C) SEC24C is not required for the colocalization of Akita with LC3B. Cells expressing Akita-sfGFP and mCherry-LC3B were depleted of SEC24C by siRNA and treated with MRT68921 for 3.5 hr. Arrowheads in the inset point to Akita puncta colocalizing with LC3B. (D) Bar graph representing the % of cells with multiple Akita-sfGFP puncta colocalizing with mCherry-LC3B puncta for the data shown in (C). Scale bars in (A–C), 10 µm. Error bars in (D) represent SEM; n = 3 independent experiments. Approximately 15–30 cells/experiment were quantitated. NS: not significant (p≥0.05); *p<0.05, **p<0.01, Student’s unpaired t-test.