Figure 4.
NME3 inhibits ER-phagy. (A) NME3 is represented as a prominent blue dot on the volcano plot. Red dots on the graph denote common regulators across all four conditions. (B) Control and NME3 KO HEK293A cells were treated with tunicamycin (10 µg/ml) for 6 h in the presence or absence of Baf. Changes in p62 and LC3B levels were analyzed using WB. The effectiveness of tunicamycin was assessed through CHOP analysis. (C) Control and KO cells were incubated with tunicamycin (10 µg/ml) for 6 h. p62 and LC3B puncta were visualized and quantified by IF. Scale bars, 10 µM. (D) Control and KO cells were treated with tunicamycin in the presence or absence of Baf. FAM134B signaling was then examined using WB. (E) Indicated cells were incubated with tunicamycin. FAM134B and LC3B puncta were visualized and quantified by IF. White arrows depict LC3B and FAM134B colocalization. Scale bars, 10 µM. Scale bars for the magnification images, 2.5 µM. (F) Control and NME3 KD HCT116 cells stably expressing the ss-RFP-GFP-KDEL reporter were incubated with tunicamycin (10 µg/ml) for 6 h in the presence or absence of Baf. ER-phagy was assessed through the processing of ss-RFP-GFP-KDEL. (G) Control and NME3 KD cells expressing ER-phagy reporter KDEL were treated with tunicamycin (10 µg/ml) for 6 h. GFP, RFP, and p62 signals were visualized and quantified by IF. Scale bars, 10 µM. (H) Control and KO HEK293A cells were incubated with either acute amino acid starvation (1.5 h, -AAa) or tunicamycin (10 µg/ml, 6 h). Whole-cell lysates were immunoblotted using the antibodies indicated. Unless otherwise indicated, experiments were performed three times. Data are represented as means ± SDs, and P values were determined by two-way ANOVA. *P ≤ 0.1; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. Source data are available for this figure: SourceData F4.

NME3 inhibits ER-phagy. (A) NME3 is represented as a prominent blue dot on the volcano plot. Red dots on the graph denote common regulators across all four conditions. (B) Control and NME3 KO HEK293A cells were treated with tunicamycin (10 µg/ml) for 6 h in the presence or absence of Baf. Changes in p62 and LC3B levels were analyzed using WB. The effectiveness of tunicamycin was assessed through CHOP analysis. (C) Control and KO cells were incubated with tunicamycin (10 µg/ml) for 6 h. p62 and LC3B puncta were visualized and quantified by IF. Scale bars, 10 µM. (D) Control and KO cells were treated with tunicamycin in the presence or absence of Baf. FAM134B signaling was then examined using WB. (E) Indicated cells were incubated with tunicamycin. FAM134B and LC3B puncta were visualized and quantified by IF. White arrows depict LC3B and FAM134B colocalization. Scale bars, 10 µM. Scale bars for the magnification images, 2.5 µM. (F) Control and NME3 KD HCT116 cells stably expressing the ss-RFP-GFP-KDEL reporter were incubated with tunicamycin (10 µg/ml) for 6 h in the presence or absence of Baf. ER-phagy was assessed through the processing of ss-RFP-GFP-KDEL. (G) Control and NME3 KD cells expressing ER-phagy reporter KDEL were treated with tunicamycin (10 µg/ml) for 6 h. GFP, RFP, and p62 signals were visualized and quantified by IF. Scale bars, 10 µM. (H) Control and KO HEK293A cells were incubated with either acute amino acid starvation (1.5 h, -AAa) or tunicamycin (10 µg/ml, 6 h). Whole-cell lysates were immunoblotted using the antibodies indicated. Unless otherwise indicated, experiments were performed three times. Data are represented as means ± SDs, and P values were determined by two-way ANOVA. *P ≤ 0.1; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. Source data are available for this figure: SourceData F4.

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