Figure S2. Additional supporting data... | Rockefeller University Press

Figure S2.

Additional supporting data related toFig. 2. (A) Ca2+ influx in HEK293T-RCaMP1h cells transfected with empty vector, VMP1, or VMP1-K404/406A was measured by flow cytometry with 8 mM CaCl2. (B) Ca2+ influx in HEK293T cells transfected with empty vector or VMP1-K404/406A was measured by flow cytometry with Fluo4/Fura Red at indicated concentrations of CaCl2. (C) The expression of STIM1 in WT and STIM1-KO HEK293T cells was examined by immunoblot. (D) ER Ca2+ and SOCE in WT and STIM1-KO HEK293T cells were measured by flow cytometry. (E) HEK293T-RCaMP1h cells were preincubated with DMSO or CM4620 for 30 min. ER Ca2+ and SOCE were measured by flow cytometry. (F) HEK293T cells were transfected with indicated plasmids. Cells were preincubated with DMSO or CM4620 for 30 min, then Ca2+ influx was measured by flow cytometry with 8 mM CaCl2. (G) ER Ca2+ and SOCE in WT and ORAI1-KO HEK293T cells were measured by flow cytometry. (H) ORAI1-KO HEK293T cells were transfected with indicated plasmids; Ca2+ influx was measured by flow cytometry with 8 mM CaCl2. (I) Strategy for screening of potential VMP1 inhibitor from a compound library targeting known ion channels/membrane transporters (HY-L011A from MCE; the information of library is available in Table S2). (J) HEK293T-RcaMP1h cells were transfected with pCMV-VMP1-K404/406A together with a BFP reporter. 24 h after transfection, cells were preincubated with each compound individually at the concentration of 20 μM for 30 min, then Ca2+ influx in BFP-high cells was measured by flow cytometry with 8 mM CaCl2. A summary of screening result is shown. EGTA was used as a positive control. (A and F) Representative plots and statistics from one of three independent experiments. (B, D, E, G, and H) Representative plots from one of three independent experiments. (C) Representative blots from one of two independent experiments. n = 5 in A, n = 3 in F; each symbol represents one sample; data represent mean ± SEM, two-way ANOVA in A and F; * <0.05, ** <0.01, *** <0.001, **** <0.001. Source data are available for this figure: SourceData FS2.

Additional supporting data related to Fig. 2 . (A) Ca²⁺ influx in HEK293T-RCaMP1h cells transfected with empty vector, VMP1, or VMP1-K404/406A was measured by flow cytometry with 8 mM CaCl₂. (B) Ca²⁺ influx in HEK293T cells transfected with empty vector or VMP1-K404/406A was measured by flow cytometry with Fluo4/Fura Red at indicated concentrations of CaCl₂. (C) The expression of STIM1 in WT and STIM1-KO HEK293T cells was examined by immunoblot. (D) ER Ca²⁺ and SOCE in WT and STIM1-KO HEK293T cells were measured by flow cytometry. (E) HEK293T-RCaMP1h cells were preincubated with DMSO or CM4620 for 30 min. ER Ca²⁺ and SOCE were measured by flow cytometry. (F) HEK293T cells were transfected with indicated plasmids. Cells were preincubated with DMSO or CM4620 for 30 min, then Ca²⁺ influx was measured by flow cytometry with 8 mM CaCl₂. (G) ER Ca²⁺ and SOCE in WT and ORAI1-KO HEK293T cells were measured by flow cytometry. (H) ORAI1-KO HEK293T cells were transfected with indicated plasmids; Ca²⁺ influx was measured by flow cytometry with 8 mM CaCl₂. (I) Strategy for screening of potential VMP1 inhibitor from a compound library targeting known ion channels/membrane transporters (HY-L011A from MCE; the information of library is available in Table S2). (J) HEK293T-RcaMP1h cells were transfected with pCMV-VMP1-K404/406A together with a BFP reporter. 24 h after transfection, cells were preincubated with each compound individually at the concentration of 20 μM for 30 min, then Ca²⁺ influx in BFP-high cells was measured by flow cytometry with 8 mM CaCl₂. A summary of screening result is shown. EGTA was used as a positive control. (A and F) Representative plots and statistics from one of three independent experiments. (B, D, E, G, and H) Representative plots from one of three independent experiments. (C) Representative blots from one of two independent experiments. n = 5 in A, n = 3 in F; each symbol represents one sample; data represent mean ± SEM, two-way ANOVA in A and F; * <0.05, ** <0.01, *** <0.001, **** <0.001. Source data are available for this figure: SourceData FS2.