Fig. 5: SMER28 enhances VCP stimulation of PI3K complex formation to induce autophagy in a Beclin 1-dependent manner. | Nature Communications

Fig. 5: SMER28 enhances VCP stimulation of PI3K complex formation to induce autophagy in a Beclin 1-dependent manner.

From: Compounds activating VCP D1 ATPase enhance both autophagic and proteasomal neurotoxic protein clearance

Fig. 5: SMER28 enhances VCP stimulation of PI3K complex formation to induce autophagy in a Beclin 1-dependent manner.

a, b Beclin 1 levels in HeLa cells treated with 20 μM SMER28 for 2, 4, or 6 hours. In the presence of 400 nM BafA1 b; n = 5. c, d Beclin 1 knockout cells and control cells were pre-treated with 400 nM BafA1 for 1 h prior to treatment with 20 μM SMER28 or 10 μM DBeQ for 8 h in the presence of BafA1. LC3-II to actin ratios are quantified in d n = 4; one-way ANOVA: P < 0.0001 with post hoc Tukey test, Ctrl SMER28 P = 0.0027, DBeQ P < 0.0001, Ctrl vs. KO P < 0.0001, KO SMER28 P = 0.0057. e Number of LC3 puncta in Beclin 1 knockout cells and control cells. Cells were pre-treated with 400 nM BafA1 for 1 h prior to treatment with 10 μM NW1030 for 8 h; n = 4; control cells P = 0.0457, Beclin 1 knockout P = 0.5854. f, g Immunoprecipitation of endogenous ATG14L from HeLa cells treated with 20 μM SMER28 or 10 μM DBeQ for 6 h. Ratios of proteins co-immunoprecipitated with ATG14L in the denoted conditions, normalized to ratios in DMSO control in g n = 7. h, i In vitro assembly of PI3K complexes. FLAG-tagged PI3K components were added individually (VPS15 and VPS34 purified and added together) and incubated alone or together with purified VCP and/or 20 μM SMER28, followed by immunoprecipitation of ATG14L. i Ratios of proteins co-immunoprecipitated with ATG14L in the denoted conditions, normalized to ratios in DMSO control. Quantification of VCP levels shows VCP/ATG14L ratios with and without the addition of SMER28, normalized to ratio without SMER28; n = 6. See also Supplementary Fig. 5c for differences in PI3K interactions with the addition of VCP with or without additional treatment with SMER28. f–i After protein transfer, PVDF membrane was cut and fragments were incubated with appropriate primary antibodies. Data presented as normalized mean ± SEM, *P < 0.05, **P < 0.001, ***P < 0.0001, one sample t test unless stated otherwise; ns not significant, Ctrl control cells. Source data are provided as a Source Data file.

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