human erbb2 her2 protein  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: ATPIF1 knockdown impairs in vitro antitumor activity and rewires the metabolism of CAR-T cells. (A) Schematic diagrams of lentiviral vector constructs for Her2 CAR-T, Her2-IF1 CAR-T, and Her2-shIF1 CAR-T cells. a U6 promoter was inserted downstream of the Her2 CAR sequence to force the shRNA of ATPIF1. (B, C) ATPIF1 protein expression in CAR-T cells analyzed by Western blotting. (D) ATPIF1 mRNA levels in CAR-T cells determined by RT-qPCR. (E) Flow cytometric analysis of CAR-positive cells: CAR-T cells were first stained with biotin-labeled Her2 ectodomain protein, followed by incubation with PE-conjugated anti-biotin antibody for FACS detection of CAR + cells, the Y-axis was the count of cell numbers. (F) In vitro proliferation of CAR-T cells. The Her2 CAR-T, Her2-IF1 CAR-T and Her2-shIF1 CAR-T cells were cultivated with IL-2 stimulation (50 IU/ml) for 12 days, the numbers of CAR + cells were determined every two days. (G-I) Basal oxygen consumption rates (OCR) of resting Her2 CAR-T, Her2-IF1 CAR-T, and Her2-shIF1 CAR-T cells. Spare Respiratory Capacity (SRC) was calculated and plotted for both resting and activated cell states. (J, M) Cytolytic activity of untransfected T cells (UNT), Her2 CAR-T, Her2-IF1 CAR-T, and Her2-shIF1 CAR-T cells against SKBR3-Luc + cells at different effector-to-target (E:T) ratios following 24-hour co-culture under 21% O 2 and 1% O 2 conditions, respectively. (K, N) IL-2 concentration in co-culture supernatants from the experiment in (J, M) , respectively. (L, O) IFN-γ concentration in co-culture supernatants from the experiment in (J, M) , respectively. The ELISA method was used to determine the concentration of IL-2 and IFN-γ. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001..

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: Knockdown, In Vitro, Activity Assay, Plasmid Preparation, Construct, Sequencing, shRNA, Expressing, Western Blot, Quantitative RT-PCR, Staining, Labeling, Incubation, Co-Culture Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: ATPIF1 overexpression enhances the in vitro antitumor activity and oxygen consumption rate (OCR) of Her2-targeted CAR-T cells. (A) Schematic diagrams of lentiviral vector constructs for Her2 CAR-T and Her2-IF1 CAR-T cells. The ATPIF1 gene is integrated in tandem after the Her2 CAR vector. (B) Flow cytometric identification of GFP-positive cells. (C) Cytolytic activity of untransfected T cells (UNT), Her2 CAR-T, and Her2-IF1 CAR-T cells against SKBR3-Luc + cells at different effector-to-target (E:T) ratios following 24-hour co-culture. Lytic function was evaluated via bioluminescent killing assay in 96-well microplates. Data are presented as mean ± standard deviation (SD) of triplicate wells from three independent experiments. (D, E) Cytokines determination. Levels of IL-2 (D) and IFN-γ (E) in co-culture supernatants, measured by enzyme-linked immunosorbent assay (ELISA). (F–H) OXPHOS determination with the Seahorse equipment. Basal OCR of resting Her2 CAR-T and Her2-IF1 CAR-T cells. Spare Respiratory Capacity (SRC) was calculated and plotted for both resting and activated cell states. **P<0.01; ***P<0.001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: Over Expression, In Vitro, Activity Assay, Plasmid Preparation, Construct, Co-Culture Assay, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: ATPIF1 overexpression enhances CAR-T cell persistence but impairs in vivo antitumor activity. (A, B) Flow cytometric quantification of GFP + Her2 CAR-T and Her2-IF1 CAR-T cells in mouse peripheral blood mononuclear cells (PBMCs) and spleens. NCG mice (n=4 in each group) were intravenously injected with Her2 CAR-T, Her2-IF1 CAR-T cells (1x10 6 CAR-T cells per mouse). Three weeks later, the mice were euthanized, the peripheral blood and spleen were collected, respectively. The flow cytometry was used to detect the percentage of GFP + CAR-T cells. (C) Tumor growth inhibition by Her2 CAR-T and Her2-IF1 CAR-T cells in SKBR-3 xenografted NCG mice. NCG mice were xenografted with SKBR3 cells (n=5 or 6 in each group). When tumors reached ~100 mm³, mice were intravenously injected with untransfected T cells (UNT), Her2 CAR-T, Her2-IF1 CAR-T cells (1x10 6 CAR-T cells per mouse). Three weeks later, the mice were euthanized, tumors were excised and weighed. *P < 0.05; **P < 0.01. (D, F) The memory phenotype of Her2 CAR-T and Her2-IF1 CAR-T cells under 21% O 2 and 1% O 2 after stimulated with the conditional medium of SKBR-3 cells. (E, G) The exhaustion markers of PD-1, LAG-3 and TIM-3 determination in Her2 CAR-T and Her2-IF1 CAR-T cells under 21% O 2 and 1% O 2 after stimulated with the conditional medium of SKBR-3 cells. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: Over Expression, In Vivo, Activity Assay, Injection, Flow Cytometry, Inhibition

Journal: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: Her2-shIF1 CAR-T cells exhibit enhanced in vivo antitumor activity. (A, B) In vivo tumor growth inhibition by UNT, Her2 CAR-T, Her2-IF1 CAR-T, and Her2-shIF1 CAR-T cells. NCG mice (n=5 or 6 in each group) were xenografted with SKBR3 cells. When tumors reached ~100 mm³, mice were intravenously injected with UNT, Her2 CAR-T, Her2-IF1 CAR-T cells (1x10 6 CAR-T cells per mouse). Two weeks later, the mice were euthanized, tumors were excised and weighed. (C) Tumor growth curves of xenografted tumors in each treatment group, the tumor size is calculated with the formula 0.5*a*b 2 , a is the length of tumor and b is the width of tumor. (D) Body weight changes of mice during the experimental period. (E) Spleen weights of mice at the end of the experiment. *P < 0.05; ***P < 0.001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: In Vivo, Activity Assay, Inhibition, Injection

Journal: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: Immunofluorescence and multiplex immunohistochemistry analyses. (A, B) Enhanced CD3 + staining in group of Her2-shIF1 CAR-T in SKBR-3 xenografted tumor tissues. NCG mice were xenografted with SKBR3 cells (n=5 in each group); when tumors reached ~100 mm³, mice were intravenously injected with untransfected T cells (UNT), Her2 CAR-T, Her2-IF1 CAR-T, or Her2-shIF1 CAR-T cells. Four days later, Mice were euthanized, and tumors were excised for immunofluorescence staining. (C) Multiplex immunohistochemistry staining of 18 formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections, showing ATPIF1 (yellow), CD3 (green), HIF-1α (orange), and DAPI (blue). (D, E) Quantitative analysis of the correlation between CD3 + staining and ATPIF1 + cells (D) , and between HIF-1α + and ATPIF1 + cells (E) from the results of Multiplex immunohistochemistry staining in (C) respectively. **P<0.01, ***P<0.001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: Immunofluorescence, Multiplex Assay, Immunohistochemistry, Staining, Injection, Formalin-fixed Paraffin-Embedded

Journal: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: ATPIF1 knockdown induces mPTP opening, mtDNA leakage, and STING signaling pathway activation. (A, B) Flow cytometric detection of mPTP opening using Calcein-AM staining. The Her2 CAR-T, Her2-IF1 CAR-T and Her-shIF1 CAR-T cells were treated with or without N-Acetylcysteine (NAC, 10 mM) for 24 hours, and then stained with Calcein-AM, flow cytometry was used to detect the content of Calcein-AM, which indicated the levels of mPTP opening. (C) Western blot analysis of p-STING, total STING, and VDAC protein expression. (D) Quantification of p-STING relative to total STING. (E) Quantification of VDAC relative to β-actin (loading control). (F–H) RT-qPCR analysis of cytoplasmic mtDNA (ND-1 and D-loop) and mRNA levels of IFI44 and IFN-β. Cytoplasmic RNA was extracted for detection. (I) Generation of ρ 0 CAR-T cells via ethidium bromide (EB) treatment for 2 weeks. Total mtDNA was extracted to detect D-loop levels. (J) RT-qPCR analysis of IFI44 mRNA expression. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: Knockdown, Activation Assay, Staining, Flow Cytometry, Western Blot, Expressing, Control, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: The enhanced in vivo antitumor activity of Her2-shIF1 CAR-T cells is dependent on STING activation. (A, B) Transwell migration assay of CAR-T cells under hypoxic conditions (1% O 2 ) with or without H151 inhibition. CAR-T cells were stained with Cell-Tracker Red CMTPX and seeded into the upper Transwell chamber. Migrated cells in the lower chamber were visualized via fluorescence microscopy, with five random fields per well imaged for quantification. (C, D) Excised tumor size and weight following treatment with different CAR-T cells, either alone or in combination with H151. NCG mice were xenografted with SKBR3 cells (n=5 in each group). When tumors reached ~100 mm³, mice were intravenously injected with untransfected T cells (UNT), Her2 CAR-T, Her2-IF1 CAR-T, or Her2-shIF1 CAR-T cells (1x10 6 CAR-T cells per mouse); or treated with or without H151 via intraperitoneal injection (7 mg/kg) as indicated. Two weeks later, the mice were euthanized, tumors were excised and weighed. **P < 0.01; ***P < 0.001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: In Vivo, Activity Assay, Activation Assay, Transwell Migration Assay, Inhibition, Staining, Fluorescence, Microscopy, Injection