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

Journal: iScience

Article Title: Localized phosphoinositide metabolism regulates STIM1/ORAI1 fast inactivation

doi: 10.1016/j.isci.2026.115543

Figure Lengend Snippet: MTMR7 double mutants and ORAI1 inactivation (A) Immunoblot analysis of HEK293T cells expressing MTMR7 and MTMR7 mutants (MTMR7-C338S, MTMR7-D343A, MTMR7-C338S + S569∗, and MTMR7-D343A + S569∗). (B) Representative whole-cell current traces of heterologously expressed ORAI1 and STIM1, along with MTMR7 double mutants; MTMR7-C338S + S569∗ or MTMR7-D343A + S569∗ in MTMR7 −/− cells. Cells were transfected in a 1:2:1 (ORAI1:STIM1:MTMR7 double mutants) ratio. Currents were recorded in 20 mM Ca 2+ during 200 ms hyperpolarizing test voltages (−60, −80, −100, −120 mV) from a 30-mV holding potential. (C) Current density (pA/pF) analysis of current from a −100-mV hyperpolarizing pulse at 3 min following whole-cell break-in (white bars; n = 7 for each group). (D) 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 = 8–9 cells for each group). For comparison, the data from wild-type MEF 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: Western Blot, Expressing, Transfection, Comparison, Control

Journal: Biomaterials Research

Article Title: A Peptide Inhibitor of Lymphocyte Activation Gene-3 Interaction with Fibrinogen-like Protein 1 Synergizes with Programmed Death-Ligand 1 Blockade to Restore T Cell Activity and Inhibit Tumor Growth

doi: 10.34133/bmr.0364

Figure Lengend Snippet: 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 MC38 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).

Article Snippet: HEK 293T (human embryonic kidney) cells, Jurkat T (human acute T cell leukemia) cells, THP-1 (acute monocytic leukemia) cells, HepG2 (human liver cancer) cells, and MC38 (C57BL/6 murine colon tumor) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Activity Assay, Isolation, Incubation, Activation Assay, Cell Culture, Staining

Journal: Biomaterials Research

Article Title: A Peptide Inhibitor of Lymphocyte Activation Gene-3 Interaction with Fibrinogen-like Protein 1 Synergizes with Programmed Death-Ligand 1 Blockade to Restore T Cell Activity and Inhibit Tumor Growth

doi: 10.34133/bmr.0364

Figure Lengend Snippet: LAG3pep-2 synergizes with anti-programmed death-ligand 1 (anti-PD-L1) antibody to inhibit tumor growth and enhance anti-tumor immunity. (A) Experimental schemes. Mice bearing subcutaneous MC38 tumor were treated with intravenous (iv) administration of LAG3pep-2 and intraperitoneal (ip) administration of anti-mouse PD-L1 and anti-mouse lymphocyte activation gene-3 (LAG-3) antibodies (10 mg/kg body weight for single treatment and 5 mg/kg body weight for combined treatment). Created with BioRender.com . (B) Tumor volumes (mm 3 ) after treatments. Data are presented as mean ± SD ( n = 5/group). * P < 0.05 ** P < 0.01 from 2-way analysis of variance (ANOVA) including all treatment groups. (C) Tumor volumes (mm 3 ) of each treatment group after tumor inoculation. (D to G) Tumor tissue cell suspensions were prepared and gated for CD45+CD3+ lymphocytes. The populations of CD4+ T cells (D), CD8+ T cells (E), and FoxP3+ T cells (F) and the ratio of CD8+ T cells/FoxP3+ T cells (G) were measured. Data are presented as mean ± SD ( n = 4/group). * P < 0.05; *** P < 0.01; ns, not significant by one-way ANOVA.

Article Snippet: HEK 293T (human embryonic kidney) cells, Jurkat T (human acute T cell leukemia) cells, THP-1 (acute monocytic leukemia) cells, HepG2 (human liver cancer) cells, and MC38 (C57BL/6 murine colon tumor) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Activation Assay

Journal: Biomaterials Research

Article Title: A Peptide Inhibitor of Lymphocyte Activation Gene-3 Interaction with Fibrinogen-like Protein 1 Synergizes with Programmed Death-Ligand 1 Blockade to Restore T Cell Activity and Inhibit Tumor Growth

doi: 10.34133/bmr.0364

Figure Lengend Snippet: LAG3pep-2 does not induce immunotoxicity in mice. (A) LAG3pep-2 was administered intraperitoneally to keyhole limpet hemocyanin (KLH)-challenged C57BL/6 mice at the indicated doses (0.01 to 100 mg/kg body weight). Cyclophosphamide (CPA) was used as a positive control. Mice were sacrificed, and spleen weights were measured on day 7 posttreatment. (B and C) Serum levels of total IgG2a (B) and total IgE (C). (D to F) The population of CD4+IFN-γ+ T cells (D), CD4+IL-4+ T cells (E), and CD4+IL-17A+ T cells (F) in the spleen. Data are presented as mean ± SD ( n = 5/group).

Article Snippet: HEK 293T (human embryonic kidney) cells, Jurkat T (human acute T cell leukemia) cells, THP-1 (acute monocytic leukemia) cells, HepG2 (human liver cancer) cells, and MC38 (C57BL/6 murine colon tumor) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Positive Control