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 promotes mitochondrial biogenesis in vivo and is associated with elevated H4K5la in ovarian GCs. (A) Schematic diagram illustrating the experimental protocol for intraperitoneal injection of FSH. In brief, mice received intraperitoneal injections of FSH (dissolved in 0.9% saline) every 12 h according to a tapered dosing regimen: 10 IU, 5 IU, and 2 doses of 2 IU, after which samples were collected. Control mice were administered an equivalent volume of 0.9% saline via intraperitoneal injection. (B) qRT-PCR examination of mitochondrial DNA ( MT-CO2 and D-Loop ) replication levels in ovarian GCs (mGCs) under the FSH dosing regimen depicted in (A). β-Actin served as the loading control for data normalization. (C) Western blot analysis of TOM20 protein levels in mGCs according to the FSH administration protocol in (A). (D) The expression level of TOM20 in (C) was determined by quantitative analysis, with TUBA1A serving as the internal control for normalization. (E) Mitochondrial morphology and quantity in mGCs were analyzed by TEM according to the FSH administration protocol in (A). Scale bar, 500 nm. (F) The ATP level in mGCs was measured after intraperitoneal injection of FSH, with normalization to the total protein concentration. (G) Immunohistochemical staining for Pan-Kla protein to assess protein levels and cellular localization in ovarian tissues according to the FSH administration protocol in (A). Scale bar, 100 μm. (H) The number of Pan-Kla + in (G) was quantified and normalized to the total number of cells. (I) Protein expression profiling via Western blot for Pan-Kla in mGCs, as per the FSH dosing regimen depicted in (A). (J) The expression level of Pan-Kla in (I) was determined by quantitative analysis, with TUBA1A serving as the internal control for normalization. (K) Collision-induced dissociation (CID) MS analysis of histone H4 modifications. The MS/MS spectrum of the peptide sequence “(Kla)SGRGKGGKGLGK” highlights the H4 lactylation site. (L) Immunohistochemical detection of H4K5la protein levels and localization in ovarian tissue according to the FSH administration protocol in (A). Scale bar, 100 μm. (M) The number of H4K5la + in (L) was quantified and normalized to the total number of cells. (N) Western blot detection of H4K5la levels in mGCs under FSH treatment (A). (O) The expression level of TOM20 in (N) was determined by quantitative analysis, with histone H4 serving as the internal control for normalization. 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: In Vivo, Injection, Saline, Control, Quantitative RT-PCR, Western Blot, Expressing, Protein Concentration, Immunohistochemical staining, Staining, Tandem Mass Spectroscopy, Sequencing

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: Suppression of lactylation inhibits FSH-induced mitochondrial biogenesis in GCs. (A) ECARs were analyzed in KGN cells with or without FSH treatment. (B) Assessment of intracellular lactate concentrations subsequent to 2 h of 15 mM 2-DG or 15 mM oxamate treatment, and then 12 h of 5-IU FSH administration. Protein concentration served as the normalization control. (C) mGCs/KGN cells after 2-h 2-DG (10 mM) and oxamate (10 mM) pretreatment and then 12-h FSH (5 IU) exposure, and Pan-Kla and H4K5la protein levels were detected by Western blot. (D) Proteins levels in (C) were quantified as follows: Pan-Kla was normalized to TUBA1A, and H4K5la was normalized to histone H4. (E) qRT-PCR assessment of mtDNA quantity ( MT-CO2 and D-Loop ) after 2-h exposure to 10 mM oxamate and subsequent 12-h treatment with 5 IU of FSH. β-Actin served as the loading control for data normalization. (F) Assessment of TOM20 protein concentrations via Western blot in mGCs and KGN cells after 2-h 10 mM oxamate exposure, and then subjected to 5 IU of FSH therapy for 12 h. (G) Protein quantification in (F) was performed using TUBA1A for normalization. (H) Following cotransfection with both LDHA siRNA and LDHB siRNA for 12 h, cells were treated with 5 IU of FSH for 12 h, after which intracellular lactate levels were measured. Protein concentration served as the normalization control. (I) mGCs and KGN cells were transfected with LDHA and LDHB siRNA for 12 h, followed by a 12-h treatment with 5 IU of FSH, after which the protein levels of Pan-Kla and H4K5la were analyzed by Western blot. (J) Proteins levels in (I) were quantified as follows: Pan-Kla was normalized to TUBA1A, and H4K5la was normalized to histone H4. (K) Analysis of mtDNA copy counts for MT-CO2 and D-Loop post-siRNA transfection (12 h), and then treated with 5 IU of FSH for another 12 h. β-Actin served as the loading control for data normalization. (L) mGCs and KGN cells were first subjected to LDHA/LDHB knockdown for 12 h and then treated with 5 IU of FSH for another 12 h, after which TOM20 protein levels were analyzed by Western blot. (M) The protein levels of TOM20 in (L) were quantitatively analyzed with normalization to TUBA1A. (N) Following a 12-h transfection of KGN cells with LDHA/LDHB siRNAs, the cells were then treated with 5 IU of FSH for another 12 h. This treatment was conducted both with and without the addition of 15 mM sodium lactate. Afterward, we assessed the protein levels of Pan-Kla and H4K5la using Western blot analysis. (O) Proteins levels in (N) were quantified as follows: Pan-Kla was normalized to TUBA1A, and H4K5la was normalized to histone H4. (P) KGN cells underwent LDHA/LDHB siRNA transfection for 12 h followed by 12-h culture with 5 IU of FSH, supplemented either with or without 15 mM sodium lactate. qRT-PCR examination of mtDNA replication gene (MT-CO2 and D-Loop) copy counts. β-Actin served as the loading control for data normalization. (Q) After transfection with LDHA/LDHB siRNA for 12 h, KGN cells were treated with 5 IU of FSH for another 12 h in medium with or without 15 mM sodium lactate. TOM20 levels were then assessed by immunoblotting. (R) The protein levels of TOM20 in (Q) were quantitatively analyzed with normalization to TUBA1A. (S) KGN cells underwent LDHA/LDHB siRNA transfection for 12 h followed by 12-h culture with 5 IU of FSH, supplemented either with or without 15 mM sodium lactate. OCRs were determined. (T) KGN cells underwent LDHA/LDHB siRNA transfection for 12 h followed by 12-h culture with 5 IU of FSH, supplemented either with or without 15 mM sodium lactate. The ATP level was measured. Protein concentration served as the normalization control. 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: Protein Concentration, Control, Western Blot, Quantitative RT-PCR, Cotransfection, Transfection, Knockdown

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 promotes mitochondrial biogenesis in GCs via P300-mediated H4K5la. (A) H4K5la levels were assessed by Western blotting after transfection with acetyltransferase siRNA for 24 h. (B) H4K5la protein levels in (A) were quantitatively analyzed with normalization to H4. (C) Western blot analysis of Pan-Kla and H4K5la protein expression in GCs and KGN treated with 10 μM C646 for 2 h, followed by 5 IU of FSH for 12 h. (D) Protein levels of Pan-Kla and H4K5la in (C) were quantified. Specifically, Pan-Kla was normalized against TUBA1A, and H4K5la was normalized against H4. (E) qRT-PCR analysis of MT-CO2 and D-Loop in KGN treated with 10 μM C646 for 2 h, followed by 5 IU of FSH for 12 h. β-Actin served as the loading control for data normalization. (F) Western blot assessing TOM20 expression in GCs and KGN exposed to 10 μM C646 (2 h) and then 5 IU of FSH (12 h). (G) The proteins levels of TOM20 in (F) were quantitatively analyzed with normalization to TUBA1A. (H) KGN cells underwent a 2-h pretreatment with 10 μM C646 and then were exposed to 5 IU of FSH for 12 h, after which OCR was evaluated. (I) Western blot detection of Pan-Kla and H4K5la levels in KGN cells after 12 h of P300 siRNA transfection and subsequent 12-h exposure to 5 IU of FSH. (J) Protein levels of Pan-Kla and H4K5la in (I) were quantified. Specifically, Pan-Kla was normalized against TUBA1A, and H4K5la was normalized against H4. (K) Western blot for Pan-Kla and H4K5la proteins in KGN cells after 12 h of CBP siRNA transfection and subsequent 12-h treatment with 5 IU of FSH. (L) Protein levels of Pan-Kla and H4K5la in (K) were quantified. Specifically, Pan-Kla was normalized against TUBA1A, and H4K5la was normalized against H4. (M) qRT-PCR evaluation of mitochondrial DNA ( MT-CO2 and D-Loop ) copies in KGN cells posttransfection with P300 or CBP siRNA for 12 h, and then subjected to 5 IU of FSH treatment for an additional 12 h. β-Actin served as the loading control for data normalization. (N) Western blot assessing TOM20 expression KGN posttransfection with P300 or CBP siRNA for 12 h, and then subjected to 5 IU of FSH treatment for an additional 12 h. (O) The proteins levels of TOM20 in (N) were quantitatively analyzed with normalization to TUBA1A. 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: Western Blot, Transfection, Expressing, Quantitative RT-PCR, Control

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