Journal: iScience
Article Title: Localized phosphoinositide metabolism regulates STIM1/ORAI1 fast inactivation
doi: 10.1016/j.isci.2026.115543
Figure Lengend Snippet: Catalytically active MTMR7 is essential for STIM1/ORAI1 activity (A) Schematic representation of the MTMR7 protein domains. The PH-GRAM domain is shown in yellow, the catalytic protein tyrosine phosphatase (PTP) domain in green, and the coiled-coil (CC) domain in pink. The location of the STIM1 binding region at the C-terminus of MTMR7 is indicated in the figure. The location of the stop codon introduced at the N-terminal amino acid residues of the STIM1 interaction site (S569∗) is indicated by a red X, the location of C338S and D343A mutations in the catalytic PTP domain is indicated by blue arrows. (B) Immunoblot analysis of wild-type and MTMR7 mutants expressed in HEK293T cells. Anti-GAPDH was used as a loading control. (C and D) Co-immunoprecipitation of MTMR7 and STIM1 in HEK293T cells co-transfected with STIM1-YFP and either MTMR7, MTMR-C338S, or MTMR-D343A mutants. Immunoprecipitation was performed using GFP-Trap agarose beads, followed by immunoblotting with anti MTMR7 ( C ) and anti STIM1 ( D ) antibodies. (E) Representative whole-cell current traces of heterologously expressed ORAI1 and STIM1, along with MTRM7 or MTMR7 mutants MTMR7-C338S, MTMR7-D343A, and MTMR7-S569∗ in MTMR7 −/− cells. Cells were transfected in a 1:2:1 (ORAI1:STIM1:MTMR7 or MTMR7 mutants) ratio. Currents were recorded in 20 mM Ca 2+ during 200 ms hyperpolarizing test voltages (−60, −80, −100, and −120 mV) from a 30-mV holding potential. (F) Current density (pA/pF) analysis of current from a −100-mV hyperpolarizing pulse at 3 min following whole-cell break-in (white bars; n = 6–13 for each group). Green bar denotes the current density measured from a −100-mV pulse at 15 min following whole-cell break-in ( n = 3). A nonparametric Kruskal-Wallis ANOVA on ranks test, followed by a multiple comparison (Dunn’s) post hoc test, was used to compare each group against either the control ( Ctrl ) condition in wild-type or MTMR7 −/− MEF cells (∗ p < 0.001 vs. wild-type, # p < 0.01 vs. MTMR7 −/− ). (G) Extent of STIM1/ORAI1 CDI from recordings of each group shown in F. Line and scatterplot summarizing the fraction of current remaining for each group, measured as the percent of peak current from the beginning and the end of the 200 ms hyperpolarizing steps. Each data point represents the mean ± SEM ( n = 6–15 cells for each group). For comparison, the data from wild-type or MTMR7 −/− cells were superimposed from D in gray and black dashed lines, respectively. A one-way ANOVA followed by a Dunnett’s post hoc test was used to compare the residual current of all groups against the control (WT) (∗ p < 0.05 at all test potentials). Data are represented as mean ± SEM.
Article Snippet: Both MEF cells and human embryonic kidney cells (HEK293T; female, ATCC Cat# MSPP-CRL3216) were cultured in accordance with established protocols using DMEM-10 (Dulbecco’s Modified Eagle’s Medium supplemented with 10% Fetal Bovine Serum, Gibco) under standard cell culture conditions of 37°C and 5% CO 2 .
Techniques: Activity Assay, Binding Assay, Western Blot, Control, Immunoprecipitation, Transfection, Comparison
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![Combined treatment with LAG3pep-2 and anti-programmed death-ligand 1 (anti-PD-L1) antibody restores T cell activity against tumor cells. (A) Experimental schemes. CD8+ T cells were isolated from the spleen of <t>MC38</t> tumor-bearing mice and incubated for 48 h with anti-CD3/CD28 beads (activation) and interleukin-2 (IL-2)/interleukin-15 (IL-15) (proliferation). The activated T cells were co-cultured with MC38 cells in the absence or presence of LAG3pep-2 and anti-mouse PD-L1 antibody alone or in combination. Created with BioRender.com . (B) Activated CD8+ T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) dye and co-cultured with tumor cells for 24 h. The population of CD3+/CFSE− cells was measured. (C to E) After co-culturing for 24 h, the culture medium was collected, and the percentage of cell death (lactate dehydrogenase [LDH] release) (C) and the concentrations of interferon-γ (IFN-γ) (D) and granzyme B (E) were measured. Data are presented as the mean ± SD of 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant by one-way analysis of variance (ANOVA).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2577/pmc13172577/pmc13172577__bmr.0364.fig.006.jpg)
