Additional file 1 of Long noncoding RNA BCRP3 stimulates VPS34 and autophagy activities to promote protein homeostasis and cell survival
Creators: name: Yan, Ruei-Liang
name: Luan, Chiu-Lin
name: Liao, Chun-Chieh
name: Liu, Li-Heng
name: Chen, Fei-Yun
name: Chen, Hsin-Yi
name: Chen, Ruey-Hwa
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1. BCRP3 expression in various human tissues. RNA-seq data from indicated tissues were retrieved from GTEx database. Fig. S2. BCRP3 desensitizes cancer cells to chemotherapeutic agent without affecting DNA damage sensing/repair. A HCT116 cells stably expressing vector or BCRP3 were treated with or without 5 μM 5-FU for 48 h, and cell viability was determined by MTT assay. The expression levels of BCRP3 were analyzed by qRT-PCR and shown on the right. Data are means ± SD from three independent experiments. P values are determined by unpaired t-test, ***P &lt; 0.001; ns, not significant. B Western blot analysis of indicated proteins from cells as in A and treated with 5 μM 5-FU for indicated time points. C HCT116 cells stably expressing BCRP3 were treated with 5 μM 5-FU for 48 h, followed by 200 nM bafilomycin A1 or 10 mM 3-MA for 2 h. Cell viability was determined by MTT assay. Data are means ± SD from three independent experiments. P values are determined by one-way ANOVA with Tukey’s post hoc test, **P &lt; 0.01, ***P &lt; 0.001. Figure S3. BCRP3 promotes autophagosome formation. A Immunofluorescence staining of LC3 in HCT116 cells stably expressing vector or BCRP3 and starved in EBSS for 2 h. Representative confocal images are shown on the left and quantitative data are on the right. Bar, 10 μm. B Immunofluorescence staining of LC3 in 293T cells stably expressing control or BCRP3 shRNAs and treated with or without 200 nM bafilomycin A1 for 2 h. Representative confocal images are shown on the left and quantitative data are on the middle. Bar, 10 μm. The expression levels of BCRP3 were analyzed by qRT-PCR and shown on the right. Data in (A), (B) are means ± SD from three independent experiments and 30 cells per group per experiment were counted. P values are determined by unpaired t-test (A) or one-way ANOVA with Tukey’s post hoc test (B), **P &lt; 0.01, ***P &lt; 0.001. C Western blot analysis of LC3 in BCRP3-deficient 293T cells treated with or without 200 nM bafilomycin A1 for 2 h. Figure S4. BCRP3 deficiency decreases WIPI2 puncta. Immunofluorescence staining of WIPI2 in control or BCRP3-deficient HeLa cells starved in EBSS for 2 h. Representative confocal images are shown on the left and quantitative data are on the right. Bar, 10 μm. Arrowheads indicate the WIPI2 puncta. Data are means ± SD from three independent experiments and 10 cells per group per experiment were counted. P values are determined by one-way ANOVA with Tukey’s post hoc test, *P &lt; 0.05. Figure S5. BCRP3 deficiency does not affect VPS34 complex abundance and integrity and Beclin 1 oligomerization. A Subunit composition of VPS34 complex in control or BCRP3-deficient HeLa cells. VPS34 complexes were immunoprecipitated and the immunocomplexes were analyzed by Western blot with indicated antibodies. B Oligomerization of Beclin 1 in BCRP3-deficient HeLa cells. Lysates from control or BCRP3-deficient HeLa cells co-transfected with HA-/FLAG-Beclin 1 were immunoprecipitated with anti-FLAG M2 beads. The interaction between HA- and FLAG-tagged Beclin 1 was analyzed by Western blot with indicated antibodies. Figure S6. BCRP3 expression and effect under different conditions. A BCRP3 expression in responses to different stresses. qRT-PCR analysis of BCRP3 levels in HeLa cells starved in EBSS, or treated with 10 μg/ml puromycin, 10 μM 17-AAG, or 10 μM 17-DMAG for the indicated time points. Data are means ± SD from three independent experiments. P values are determined by one-way ANOVA with Tukey’s post hoc test; ns, not significant. B Western blot analysis using control or BCRP3-deficient HeLa cells cultured in normal conditions. Ubiquitinated protein levels were quantified and shown on the bottom. Table S1. Antibody details. Table S2. Sequences of PCR primers. Table S3. List of proteins accumulated in BCRP3-deficient cells under proteotoxicity.