Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Metformin drives HIF-1α–mediated dual metabolic reprogramming to enhance γδ T cell therapy in triple-negative breast cancer

doi: 10.1007/s00262-026-04351-w

Figure Lengend Snippet: Metformin exerts distinct effects on OXPHOS and glycolysis in γδ T cells versus TNBC cells through HIF-1α. A , B OCR profiles of TNBC cells and γδ T cells following 24-h metformin treatment, measured by Seahorse XF metabolic flux analysis. OCR curves were assessed under basal conditions and after adding the indicated mitochondrial inhibitors: oligomycin, FCCP, and rotenone/antimycin. C , D ECAR of TNBC cells and γδ T cells were quantified after 24 h of metformin exposure, with measurements taken during basal states and following sequential administration of glucose, oligomycin, and 2-DG. E Representative immunoblots showing AMPK signaling in TNBC and γδ T cells after 4 h exposure to increasing metformin doses. β-actin was employed for normalization. F , G OCR and ECAR profiles of γδ T cells following 24-h metformin and PX-478 treatment, measured by Seahorse XF metabolic flux analysis. All experimental procedures included a minimum of three independent biological replicates. Data represent mean ± SEM; statistical significance determined by two-tailed Student’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. PX-478: HIF-1α Inhibitor

Article Snippet: The membrane was blocked with 5% non-fat milk in TBST (JXF003, Jingxin Bio) for 1 h at room temperature and subsequently incubated overnight at 4 °C with primary antibodies targeting phospho-AMPKα (2535 T, CST), AMPKα (2793S, CST), HIF-1α (2178S, CST), and β-actin (4970S, CST).

Techniques: Western Blot, Two Tailed Test

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: FSH-induced deacetylation of PGC-1α by HDAC4 promotes mitochondrial biogenesis in GCs. (A) Conservation analysis of the PGC-1α K329/330 acetylation site across different species. (B) Analysis by co-IP reveals the engagement of PGC-1α with acetylated lysines, as assessed post-10 μM C646 administration for 2 h and then subjected to 5 IU of FSH exposure for 12 h in KGN cells. (C) Assessment of PGC-1α acetylation levels quantitatively in (B). The level of acetylation was calculated as the ratio of the pan-acetylated-lysine signal to the total PGC-1α signal following co-IP. (D) IP analysis identified the association of PGC-1α with pan-acetylated lysines post-HDAC4 silencing for 12 h, subsequent to 12 h of 5-IU FSH treatment in KGN cells. (E) Quantitative analysis of the acetylation modification level of PGC-1α in (D). The level of acetylation was calculated as the ratio of the pan-acetylated-lysine signal to the total PGC-1α signal following co-IP. (F) IP technique to identify the association of PGC-1α with all acetylated lysines in PGC-1α knockdown KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α (acetylation-resistant), or K329/330Q PGC-1α (acetylation-mimic) for 12 h, and then treated with 5 IU of FSH for 12 h. (G) Quantitative analysis of the acetylation modification level of PGC-1α in (F). The level of acetylation was calculated as the ratio of the pan-acetylated-lysine signal to the total Flag-PGC-1α signal following co-IP. (H) qRT-PCR analysis of mitochondrial DNA copy number ( MT-CO2 and D-Loop ) in PGC-1α knockdown KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α (acetylation-resistant), or K329/330Q PGC-1α (acetylation-mimic) for 12 h. Following transfection, the cells were stimulated with 5 IU of FSH for an additional 12-h period before analysis. β-Actin served as the loading control for data normalization. (I) Western blot analysis of TOM20 protein levels in KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α, or K329/330Q PGC-1α for 12 h, followed by treatment of 5 IU of FSH. (J) The protein levels of TOM20 in (I) were quantitatively analyzed with normalization to TUBA1A. (K) PGC-1α knockdown KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α (acetylation-resistant), or K329/330Q PGC-1α (acetylation-mimic) for 12 h, followed by 5 IU of FSH for an additional 12 h. MitoTracker Green (green) labeled mitochondria, visualized via laser confocal microscopy. Scale bar, 5 μm. (L) Quantitative analysis of MitoTracker Green fluorescence intensity from (K). (M) PGC-1α knockdown KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α (acetylation-resistant), or K329/330Q PGC-1α (acetylation-mimic) for 12 h, followed by 5 IU of FSH for an additional 12 h. The OCRs were then measured. Data are presented as the mean ± SD from at least 3 independent experiments ( n ≥ 3). Statistical differences between groups were compared by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Co-Immunoprecipitation Assay, Modification, Knockdown, Quantitative RT-PCR, Transfection, Control, Western Blot, Labeling, Confocal Microscopy, Fluorescence

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: Deacetylation of PGC-1α enhances its interaction with NRF1/2. (A) Analysis of the interaction between PGC-1α and NRF1/2 by IP in KGN cells. Cells were first treated with 15 mM oxamate for 2 h, followed by stimulation with 5 IU of FSH for 12 h. (B) Quantitative measurement of the binding affinity between PGC-1α and proteins NRF1/NRF2 in (A). The affinity is presented as the level of NRF1 or NRF2 co-immunoprecipitated with PGC-1α, normalized to the total PGC-1α protein level. (C) Co-IP assays examining PGC-1α binding to NRF1/2 within KGN cells: samples pretreated with 10 μM C646 (2 h) and then stimulated with FSH (12 h). (D) Quantitative measurement of the binding affinity between PGC-1α and proteins NRF1/NRF2 in (C). The affinity is presented as the level of NRF1 or NRF2 co-immunoprecipitated with PGC-1α, normalized to the total PGC-1α protein level. (E) Co-IP assay assessing PGC-1α and NRF1/2 binding in KGN cells post-HDAC4 knockdown (12 h) and FSH exposure (5 IU, 12 h). (F) Quantitative measurement of the binding affinity between PGC-1α and proteins NRF1/NRF2 in (E). The affinity is presented as the level of NRF1 or NRF2 co-immunoprecipitated with PGC-1α, normalized to the total PGC-1α protein level. (G) Co-IP analysis of PGC-1α/NRF1/2 binding dynamics in KGN cells expressing Flag-tagged WT, K329/330R (acetylation-resistant), or K329/330Q (acetylation-mimic) PGC-1α, treated with or without 5 IU of FSH for 12 h. (H) Quantitative measurement of the binding affinity between PGC-1α and proteins NRF1/NRF2 in (G). The affinity is presented as the level of NRF1 or NRF2 co-immunoprecipitated with PGC-1α, normalized to the total PGC-1α protein level. (I) KGN cells were first transfected with PGC-1α siRNA for 12 h, followed by overexpression of Flag-tagged constructs: WT PGC-1α, acetylation-resistant K329/330R PGC-1α, or acetylation-mimetic K329/330Q PGC-1α for 12 h, and then treated with 5 IU of FSH for an additional 12 h. ChIP analysis of the binding of Flag-tagged PGC-1α to the promoters of Tfb1m , Tfb2m , and Tfam. (J) KGN cells overexpressing Flag-tagged WT PGC-1α plasmid for 12 h were sequentially treated with 15 μM LMK-235 (2 h) followed by 5 IU of FSH (12 h). Subcellular fractionation was then performed to obtain cytosolic and nuclear extracts, which were subjected to immunoblot analysis using antibodies against Flag (transgene expression), TUBA1A (cytosolic marker), and histone H4 (nuclear marker). (K) PGC-1α levels in the nuclear and cytoplasmic fractions were quantified in (J). H4 and TUBA1A were used as internal controls for normalizing the nuclear and cytoplasmic proteins, respectively. (L) Immunoblot analysis was performed to examine Flag-tagged WT PGC-1α expression and subcellular localization in HDAC4-knockdown KGN cells. After 12-h Flag-PGC-1α induction, cells received 5 IU of FSH for another 12 h, and cytosolic and nuclear fractions were probed for Flag, TUBA1A (cytosolic marker), and histone H3 (nuclear marker). (M) Quantitatively measure the subcellular distribution of PGC-1α between the nucleus and cytoplasm in (L). H4 and TUBA1A were used as internal controls for normalizing the nuclear and cytoplasmic proteins, respectively. (N) KGN cells were first transfected with PGC-1α siRNA for 12 h, followed by overexpression of Flag-tagged constructs: WT PGC-1α, acetylation-resistant K329/330R PGC-1α, or acetylation-mimetic K329/330Q PGC-1α for 12 h, and then treated with 5 IU of FSH for an additional 12 h. (O) Quantitatively measure the subcellular distribution of PGC-1α between the nucleus and cytoplasm in (N). H4 and TUBA1A were used as internal controls for normalizing the nuclear and cytoplasmic proteins, respectively. (P) Immunofluorescence analysis of PGC-1α subcellular localization in KGN cells transfected with Flag-tagged WT, K329/330R, or K329/330Q PGC-1α for 12 h, followed by treatment 5 IU of FSH for 12 h. (Q) Quantitative analysis of Flag fluorescence intensity from (P). Data are presented as the mean ± SD from at least 3 independent experiments ( n ≥ 3). Statistical differences between groups were compared by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Knockdown, Expressing, Transfection, Over Expression, Construct, Plasmid Preparation, Fractionation, Western Blot, Marker, Immunofluorescence, Fluorescence

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: C646-mediated P300 inhibition inhibits mitochondrial biogenesis and follicular development in vivo. (A) Schematic diagram of the in vivo experimental procedure. Mice were randomly assigned to 5 groups: (1) control (DMSO/0.9% saline vehicle), (2) FSH alone, (3) FSH + C646 (15 mg/kg), (4) FSH + LMK-235 (15 mg/kg), and (5) FSH + SR-18292 (15 mg/kg). All intraperitoneal injections were administered at 12-h intervals. The FSH regimen followed a tapering protocol of 10 IU, 5 IU, and two 2-IU doses. The respective inhibitors were co-administered with each FSH injection. All drugs were dissolved in DMSO and diluted in 0.9% saline for administration. (B) Western blot analysis of Pan-Kla within histone regions and H4K5la levels following the indicated treatments in (A), with H4 used as a loading control for normalization. (C) Immunohistochemical detection of Pan-Kla expression following the indicated treatments in (A). Pan-Kla + normalized to total cell number. Scale bar, 200 μm. (D) qRT-PCR measurement of HDAC4 expression after specified treatments in (A). Tuba1a served as the loading control for data normalization. (E) Western blot assessment of HDAC4 expression posttreatment in (A), with TUBA1A used as a loading control for normalization. (F) Co-IP assay assessing PGC-1α and pan-acetyl-lysine binding posttreatment in (A). For IP, PGC-1α acetylation was quantified as the ratio of acetylated to total PGC-1α. For Input, the levels of total acetylation and PGC-1α protein were normalized to TUBA1A. (G) Co-IP assay assessing PGC-1α and NRF1/2 binding posttreatment in (A). For IP, the binding of PGC-1α to NRF1/2 was measured by calculating the NRF1/2 to PGC-1α ratio. For Input, the levels of NRF1/2 and PGC-1α were normalized to TUBA1A. (H) qRT-PCR examination of Tfb1m , Tfb2m , and Tfam mRNA expression after the specified treatments in (A). Tuba1a served as the loading control for data normalization. (I) qRT-PCR was used to assess mitochondrial DNA copy number, specifically targeting the MT-CO2 and D-Loop regions, following the indicated treatments in (A). β-Actin served as the loading control for data normalization. (J) Western blot assessment of TOM20 expression posttreatment in (A), with TUBA1A used as a loading control for normalization. (K) Using a radioimmunoassay (RIA), we quantified the serum estradiol (E2) concentrations across the treatment groups specified in (A). (L) Western blot assessment of CYP19A1 expression posttreatment in (A), with TUBA1A used as a loading control for normalization. (M) Western blot assessment of proliferating cell nuclear antigen (PCNA) expression posttreatment in (A), with TUBA1A used as a loading control for normalization. (N) 5-Ethynyl-2’-deoxyuridine (EdU) incorporation assay detects the proliferation activity of mouse ovarian GCs following the indicated treatments in (A). EdU-positive cells normalized to total cell number. Scale bar, 100 μm. (O) Measurement of ovarian size following the indicated treatments in (A). (P) Measurement of ovarian weight following the indicated treatments in (A). The ovary weight was expressed relative to the body weight of the corresponding mouse. (Q) Measurement of follicle diameter following the indicated treatments in (A). (R) The counts of primary, secondary, and antral follicles were assessed via hematoxylin and eosin (H&E) staining as outlined in treatment (A). PF, primary follicle; SF, secondary follicle; AF, antral follicles. Scale bar, 500 μm. Data are presented as the mean ± SD from at least 3 independent experiments ( n ≥ 3). Statistical differences between groups were compared by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Inhibition, In Vivo, Control, Saline, Injection, Western Blot, Immunohistochemical staining, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Binding Assay, RIA Assay, Activity Assay, Staining

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: Mechanistic model of H4K5la in FSH-driven mitochondrial biogenesis. FSH activates aerobic glycolysis in GCs, generating lactate that induces histone H4K5la via P300/CBP. This epigenetic modification promotes HDAC4 transcription, leading to HDAC4-mediated deacetylation of PGC-1α at K329/330. Deacetylated PGC-1α facilitates the recruitment of nuclear respiratory factors (NRF1 and NRF2) to promoter regions, initiating the expression of essential genes involved in mitochondrial biogenesis, such as TFAM , TFB1M , and TFB2M . This process ultimately leads to an expansion of the mitochondrial network. Through this lactate–H4K5la–HDAC4 axis, FSH synchronizes mitochondrial expansion with the bioenergetic demands of follicular development.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Modification, Expressing