caused defects of autophagic flux in HeLa cells. (A) Wildtype, not an inactive mutant, TPC2 expression markedly induced GFP-LC3 puncta in HeLa cells.
FIGURE LEGENDS FOR SUPPLEMENTAL FIGURES Figure S1. Starvation induced autophagy whereas bafilomycin or TPC2 overexpression caused defects of autophagic flux in HeLa cells. (A) Wildtype, not an inactive mutant, TPC2 expression markedly induced GFP-LC3 puncta in HeLa cells. Scale bar = 20 µm. (B) TPC2 overexpression or bafilomycin (1h) treatment induced the accumulation of GFP-LC3-II in GFP-LC3 expressing HeLa cells. (C) Expressions of TPC2 mRNAs in HeLa cells and MCF-7 cells were determined by qRT-PCR using two distinctive human TPC2 primers.
Figure S2. Effects of ATG5 knockdown on autophagic flux in TPC2 overexpressing HeLa cells. (A) ATG5 knockdown in HeLa cells. (B) TPC2 overexpression failed to induce accumulation of LC3-II puncta in ATG5 knockdown HeLa cells. Quantification of LC3 puncta is expressed as mean ± S.E., n= ~ 50 cells. The * symbols indicate the results of t Test analysis, p < 0.05. Scale bar = 10 µm.
Figure S3. Effects of NAADP-AM and Ned-19 on autophagic flux in TPC2 overexpressing HeLa cells. (A) NAADP-AM (100 nM) transiently increased the accumulation of both LC3-II and p62 in TPC2 overexpressing HeLa cells. (B) Long exposure of LC3 immunoblot shown in Figure 3E. (C) Ned19 (10 µM) transiently decreased the levels of both LC3-II and p62 in TPC2 overexpressing HeLa cells.
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Figure S4. TPC2 signaling inhibited the progression of autophagy during neural differentiation of mouse ES cells. (A) In vitro neural differentiation of mouse ES cells initiated by monolayer adherent culture markedly induced LC3-II in Sox1-GFP 46C mouse ES cells. (B) Expressions of TPC2 mRNAs during neural differentiation of ES cells were determined by qRT-PCR. (C) and (D) TPC2 knockdown decreased endogenous LC3-II levels (C) and LC3-II puncta (D) in D3 mouse ES cells during an in vitro neural differentiation initiated by monolayer adherent culture. (E) and (F) TPC2 knockdown decreased endogenous LC3-II puncta (E) and LC3-II levels (F) in Sox1-GFP 46C mouse ES cells during an in vitro neural differentiation initiated by monolayer adherent culture. Scale bar = 10 µm. (G) TPC2 knockdown decreased LC3-II levels during in vitro neural differentiation of D3 mouse ES cells, which was reversed by bafilomycin treatment (1 h). (H) TPC2 overexpression increased LC3-II levels during in vitro neural differentiation of D3 mouse ES cells.
Figure S5. Effects of NAADP/TPC2 signaling on lysosomal pH in HeLa cells. (A) The 3 h, not 1 h, of bafilomycin (10 nM) treatment induced the accumulation of both LC3-II and p62 in TPC2 overexpressing HeLa cell. (B) The 3 h of bafilomycin treatment induced the accumulation of both LC3-II and p62 in control, TPC2 overexpressing, and TPC2 mutant expressing HeLa cells. (C) FACS analyses of Lysosensor DND-189 staining in control or
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TPC2 overexpressing HeLa cells treated with or without bafilomycin for 3 h. (D) Microplate reader measurement of Lysosensor DND-189 stained HeLa cells. The data are expressed as mean ± S.D., n = 3. The * symbols indicate the results of t Test analysis, p < 0.05. (E) Standard curve of lysosomal pH was determined by dual-emission ratio imaging of LysoSensor Yellow/Blue DND-160 stained HeLa cells. (F) NAADP-AM transiently increased lysosomal pH in TPC2 overexpressing HeLa cells as determined by microplate reader measurement of Lysosensor DND-189 stained cells.
Figure S6. TPC2 did not inhibit general endosomal-lysosomal degradation in HeLa cells but prevent the recruitment of Rab-7 to autophagosomes. (A) TPC2 overexpression failed to change EGF-induced EGFR degradation, whereas BAF (10 nM) markedly inhibited it in HeLa cells. (B) TPC2 overexpression did not inhibit the degradation of DQ-BSA-green in HeLa cells, while BAF (10 nM) markedly inhibited it after 3 hr of BSA labeling.
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SUPPLEMENTAL TABLE 1 1. GAPDH: Forward, 5’ CTAGAGAGCTGACAGTGGGTAT; Reverse, 5’AGACGACCAATGCGTCCAAA 2. TPC2 (pair 1), Forward, 5’ CTGGTTTCGGAGATTTGCA; Reverse, 5’ CGCAGGGATCATCACATCA 3. TPC2 (pair 2), Forward, 5’ ATACGGTGGTCGCTACCAGA; Reverse, 5’CGCAGGGATCATCACATCA 4. TPC2 shRNA1, Forward, 5’ CCGGCCCAACAACTTTGACGACTTTCTCGAGAAAGTCGTCAAAGTTGTTGGGTTTTTG; Reverse, 5’ AATTCAAAAACCCAACAACTTTGACGACTTTCTCGAGAAAGTCGTCAAAGTTGTTGGG 5. TPC2 shRNA2, Forward, 5’ CCGGAACCTCTTGTCTATTTGTGTGCTCGAGCACACAAATAGACAAGAGGTTTTTTTG; Reverse, 5’ AATTCAAAAAAACCTCTTGTCTATTTGTGTGCTCGAGCACACAAATAGACAAGAGGTT 6. TPC2 shRNA3, Forward, 5’ CCGGCGACTGATGAACACACTGATTCTCGAGAATCAGTGTGTTCATCAGTCGTTTTTG; Reverse, 5’
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AATTCAAAAACGACTGATGAACACACTGATTCTCGAGAATCAGTGTGTTCATCAGTCG 7. Scramble shRNA, Forward, 5’ CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTG; Reverse, 5’ AATTCAAAAACCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG
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