α tubulin  (Proteintech)

Journal: Biochemistry and Biophysics Reports

Article Title: Normal spermatogenesis and fertility in Spmip8 deficiency male mice

doi: 10.1016/j.bbrep.2025.102406

Figure Lengend Snippet: Generation and validation of Spmip8 knockout mice. (A) Schematic diagram of Spmip8 −/− mouse creation. (B) Sanger sequencing of genomic DNA shows a deletion in the Spmip8 - gene. (C) Spmip8 −/− mice were identified by genomic PCR. (D) Spmip8 - transcripts were not detected in adult Spmip8 −/− testes, n = 3 for each genotype. (E) Western blot analysis the SPMIP8 protein in Spmip8 knockout mice. α-TUBULIN was used as a loading control. (F) Immunofluorescence staining of SPMIP8 (green), PNA (acrosome, red) in testis sections from 10-week-old WT and Spmip8 −/− mice. Magnification ×40 in the panels. DAPI (blue) stains the nuclei. The head signal in elongating spermatids is non-specific, as it appears in both WT and Spmip8 −/− testis sections. Scale bar: 50 μm ∗∗∗ P < 0.001.

Article Snippet: The membrane was blocked with 5 % nonfat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies against SPMIP8 (1:1000, HPA062092, Sigma, Germany) and α-TUBULIN (1:5000, 11224-1-AP, Proteintech, China).

Techniques: Biomarker Discovery, Knock-Out, Sequencing, Western Blot, Control, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: Genetic suppression of GCN2 reverses developmental delay and seizure phenotypes in dPARS2-deficient flies. (A) Western blot analysis of P-GCN2, GCN2 and P-PERK in protein extracts from control and elav- Gal4-driven dPARS2 knockdown fly heads. α-tubulin was used as a loading control. (B) Quantification of the Western blots shown in A. P-GCN2, N = 3; GCN2 and P-PERK, N = 4. ∗∗p < 0.01, ns, not significant. (C) Western blot analysis of P-eIF2α in protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown fly heads. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 3. ∗p < 0.05, ∗∗p < 0.01. (E) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown fly heads. Flies were fed with puromycin. α-actin was used as the loading control. (F) Quantification of the Western blots shown in E. N = 3, ∗p < 0.05, ∗∗∗p < 0.001. (G) Images of control, elav -Gal4-driven dPARS2 knockdown and elav -Gal4-driven dPARS2 and GCN2 double knockdown flies at different developmental stages. Scale bars: 500 μm. (H) Graph showing pupariation rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown larvae. N = 3, n = 28–30. (I) Graph showing eclosion rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown pupae. N = 3, n = 28–30. (J) Graph showing percentage of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies displaying Bang-sensitive paralytic phenotypes. N = 3, n = 10 sample. ∗∗∗∗p < 0.0001. (K) Graph showing the recovery time of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies from paralysis. n = 30. ∗∗∗∗p < 0.0001.

Article Snippet: Primary antibodies used were anti-MT-ND1 (Abcam, AB181848-1001), anti-MT- CO2 (Proteintech, 55070-1-AP), anti-MT-ATP8 (Proteintech, 26723-1-AP),anti-NDUFS1 (Proteintech, 12444-1-AP), anti-NDUFS3 (Abcam, ab14711), anti-UQCRFS1 (Abcam, ab14746), anti-ATP5A (Abcam, ab14748), anti-SDHB (Proteintech, 10620-1-AP), anti-Porin/VDAC (Abcam, ab14734), anti-P-eIF2α (Cell Signaling Technology, 3398), anti-eIF2α (Cell Signaling Technology, 2103), anti-P-PERK (ABclonal, AP0886), anti-PERK (ABclonal, A27664 ), anti-P-GCN2 (Abcam, ab75836), anti-GCN2 (ABclonal, A2307), anti-LDH (ThermoFisher, PA5-26531), anti-PARS2 (ABclonal, A16512), anti-His (yeasen, 30405ES50), anti-ATF4 (Abcam, ab1371), anti-Alpha actin (Proteintech, 23660-1-AP) and anti-Alpha tubulin (Proteintech, 66031-1-Ig).

Techniques: Western Blot, Control, Knockdown, Staining

Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: PARS2 V95I mutation causes mitochondrial dysfunction and ISR activation in human cells (A) Western blot analysis of ectopically expressed PARS2 proteins. Lysates from HEK-293T cells transfected with plasmids encoding His-tagged wild-type (WT) or the indicated PARS2 variants were immunoblotted with an anti-His antibody. α-actin was used as a loading control. (B) Quantification of the Western blots shown in A. N = 5, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Western blot analysis of endogenous PARS2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 4, ∗∗∗p < 0.001. (E) Western blot analysis of mtDNA-encoded CO2 and ATP8 and nuclear-DNA encoded NDUFS1, NDUFS3, UQCRFS1 and ATP5A in protein extracts from the wild-type controls and the PARS2 V95I cells. VDAC was used as a loading control. (F) Quantification of the Western blots shown in E. MT-CO2, MT-ATP8, NDUFS1, NDUFS3, and ATP5A, N = 4; UQCRFS1, N = 7. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, not significant. (G) CI, CII and CIV in-gel activity analysis of isolated mitochondria from the wild-type controls and the PARS2 V95I cells. (H) Western blot analysis of P-eIF2α and eIF2α in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (I) Quantification of the Western blots shown in H. N = 5, ∗∗∗∗p < 0.0001. (J) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as the loading control. (K) Quantification of the Western blots shown in J. N = 4. ∗∗∗∗p < 0.0001. (L) Western blot analysis of ATF4 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (M) Quantification of the Western blots shown in L. N = 5, ∗∗∗p < 0.001. (N) Western blot analysis of P-GCN2 and GCN2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (O) Quantification of the Western blots shown in N. N = 4, ∗∗p < 0.01. (P) Western blot analysis of P-PERK and PERK in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (Q) Quantification of the Western blots shown in P. N = 5, ns, not significant.

Article Snippet: Primary antibodies used were anti-MT-ND1 (Abcam, AB181848-1001), anti-MT- CO2 (Proteintech, 55070-1-AP), anti-MT-ATP8 (Proteintech, 26723-1-AP),anti-NDUFS1 (Proteintech, 12444-1-AP), anti-NDUFS3 (Abcam, ab14711), anti-UQCRFS1 (Abcam, ab14746), anti-ATP5A (Abcam, ab14748), anti-SDHB (Proteintech, 10620-1-AP), anti-Porin/VDAC (Abcam, ab14734), anti-P-eIF2α (Cell Signaling Technology, 3398), anti-eIF2α (Cell Signaling Technology, 2103), anti-P-PERK (ABclonal, AP0886), anti-PERK (ABclonal, A27664 ), anti-P-GCN2 (Abcam, ab75836), anti-GCN2 (ABclonal, A2307), anti-LDH (ThermoFisher, PA5-26531), anti-PARS2 (ABclonal, A16512), anti-His (yeasen, 30405ES50), anti-ATF4 (Abcam, ab1371), anti-Alpha actin (Proteintech, 23660-1-AP) and anti-Alpha tubulin (Proteintech, 66031-1-Ig).

Techniques: Mutagenesis, Activation Assay, Western Blot, Transfection, Control, Activity Assay, Isolation, Staining

Journal: Biomedical Reports

Article Title: Rabeprazole attenuates fibrosis by modulating SMAD3 linker region phosphorylation

doi: 10.3892/br.2025.2098

Figure Lengend Snippet: Rabeprazole modulates SMAD3 phosphorylation and nuclear translocation. (A) GES-1 and AGS cells were treated with or without rabeprazole for 1 h, and the phosphorylation of SMAD3 linker was detected by immunoblotting. (B-E) The band intensities were quantified and analyzed by one sample t-test. Data are shown as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001, n=3. (F) Left panel: The subcellular fraction was isolated using nuclear and cytoplasmic protein extraction kit according to manufacturer's instructions. The SMAD3 level was detected by western blotting, α-tubulin and lamin A/C were used as cytosolic and nuclear internal controls. Right panel: IF analysis of SMAD3 in AGS cells treated with or without rabeprazole for 1 h. Scale bar, 100 µm. SMAD3, SMAD family member 3; IF, immunofluorescence; phospho, phosphorylated.

Article Snippet: Antibodies including α-SMA specific monoclonal antibody (mAb) (cat. no. 67735-1-Ig), FN mAb (cat. no. 66042-1-Ig), vimentin polyclonal antibody (pAb) (cat. no. 10366-1-AP), collagen type I (Col1a1) mAb (cat. no. 67288-1-Ig), SMAD3 mAb (cat. no. 66516-1-Ig), lamin A/C pAb (cat. no. 10298-1-AP) and α-tubulin mAb (cat. no. 66031-1-Ig) were purchased from Proteintech Group, Inc. TIF1γ mouse mAb (cat. no. YM1108), SMAD3 (phospho Ser204) rabbit pAb (cat. no. YP0363), SMAD3 (phospho Ser213) rabbit pAb (cat. no. YP0364), SMAD3 (phospho Thr179) rabbit pAb (cat. no. YP0745) and SMAD3 (phospho Ser208) rabbit pAb (cat. no. YP0746) were purchased from Immunoway Biotechnology Co., Ltd.; peroxidase affiniPureTM goat anti-rabbit IgG (H+L) (cat. no. 111-035-003) and peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) (cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Isolation, Protein Extraction, Immunofluorescence

Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: Genetic suppression of GCN2 reverses developmental delay and seizure phenotypes in dPARS2-deficient flies. (A) Western blot analysis of P-GCN2, GCN2 and P-PERK in protein extracts from control and elav- Gal4-driven dPARS2 knockdown fly heads. α-tubulin was used as a loading control. (B) Quantification of the Western blots shown in A. P-GCN2, N = 3; GCN2 and P-PERK, N = 4. ∗∗p < 0.01, ns, not significant. (C) Western blot analysis of P-eIF2α in protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown fly heads. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 3. ∗p < 0.05, ∗∗p < 0.01. (E) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown fly heads. Flies were fed with puromycin. α-actin was used as the loading control. (F) Quantification of the Western blots shown in E. N = 3, ∗p < 0.05, ∗∗∗p < 0.001. (G) Images of control, elav -Gal4-driven dPARS2 knockdown and elav -Gal4-driven dPARS2 and GCN2 double knockdown flies at different developmental stages. Scale bars: 500 μm. (H) Graph showing pupariation rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown larvae. N = 3, n = 28–30. (I) Graph showing eclosion rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown pupae. N = 3, n = 28–30. (J) Graph showing percentage of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies displaying Bang-sensitive paralytic phenotypes. N = 3, n = 10 sample. ∗∗∗∗p < 0.0001. (K) Graph showing the recovery time of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies from paralysis. n = 30. ∗∗∗∗p < 0.0001.

Article Snippet: Mouse anti-Alpha tubulin , Proteintech , 66031-1-Ig.

Techniques: Western Blot, Control, Knockdown, Staining

Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: PARS2 V95I mutation causes mitochondrial dysfunction and ISR activation in human cells (A) Western blot analysis of ectopically expressed PARS2 proteins. Lysates from HEK-293T cells transfected with plasmids encoding His-tagged wild-type (WT) or the indicated PARS2 variants were immunoblotted with an anti-His antibody. α-actin was used as a loading control. (B) Quantification of the Western blots shown in A. N = 5, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Western blot analysis of endogenous PARS2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 4, ∗∗∗p < 0.001. (E) Western blot analysis of mtDNA-encoded CO2 and ATP8 and nuclear-DNA encoded NDUFS1, NDUFS3, UQCRFS1 and ATP5A in protein extracts from the wild-type controls and the PARS2 V95I cells. VDAC was used as a loading control. (F) Quantification of the Western blots shown in E. MT-CO2, MT-ATP8, NDUFS1, NDUFS3, and ATP5A, N = 4; UQCRFS1, N = 7. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, not significant. (G) CI, CII and CIV in-gel activity analysis of isolated mitochondria from the wild-type controls and the PARS2 V95I cells. (H) Western blot analysis of P-eIF2α and eIF2α in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (I) Quantification of the Western blots shown in H. N = 5, ∗∗∗∗p < 0.0001. (J) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as the loading control. (K) Quantification of the Western blots shown in J. N = 4. ∗∗∗∗p < 0.0001. (L) Western blot analysis of ATF4 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (M) Quantification of the Western blots shown in L. N = 5, ∗∗∗p < 0.001. (N) Western blot analysis of P-GCN2 and GCN2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (O) Quantification of the Western blots shown in N. N = 4, ∗∗p < 0.01. (P) Western blot analysis of P-PERK and PERK in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (Q) Quantification of the Western blots shown in P. N = 5, ns, not significant.

Article Snippet: Mouse anti-Alpha tubulin , Proteintech , 66031-1-Ig.

Techniques: Mutagenesis, Activation Assay, Western Blot, Transfection, Control, Activity Assay, Isolation, Staining

Journal: iScience

Article Title: HMOX1 drives dihydroartemisinin-sensitized ferroptosis antagonized by mitochondrial fusion

doi: 10.1016/j.isci.2025.114382

Figure Lengend Snippet: DHA increases sensitivity to ferroptosis by modulating cellular oxidative stress (A–E) Western blot and quantifications of the GPX4, ACSL4, FSP1, DHODH, β-actin, and α-tubulin expression in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h. (F) GSH levels in N27 cells treated with different doses of DHA (0, 1.5 μM, 12.5 μM) for 12 h were detected, n = 6 wells from one representative of two independent experiments. (G) Western blot and quantifications of the 4-HNE and β-actin expression in N27 cells treated with different doses of DHA (0, 1.5, 12.5, and 25 μM) for 12 h. (H) N27 cells were treated with different doses of DHA (0, 1.5, and 12.5 μM) for 12 h and lipid ROS was detected by C11-BODIPY using flow cytometry. Representative histograms for fluorescence of oxidized C11-BODIPY and the ratio of the MFI of oxidized to reduced C11-BODIPY are shown. (I) TEM of N27 cells treated with DHA (12.5 μM) for 12 h. Red arrows indicate shrunken mitochondria. Scale bars: upper panel = 2 μm; lower panel = 500 nm, as indicated. (J) The average fluorescence intensity of FerroOrange in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h, n = 5 wells from one representative of two independent experiments. (K) The iron levels detected by ICP-MS in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h, n = 5 wells from one representative of two independent experiments. (L) Cell viability of N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) in the absence or presence of DFO (50 μM) for 48 h, n = 6 wells from one representative of two independent experiments. (M) Cell viability of N27 cells treated with DHA (12.5 μM) in the absence or presence of DFO (50 μM) for 48 h, n = 6 wells from one representative of two independent experiments. (N) The representative images of H 2 DCFDA staining in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h, and the average fluorescence intensity are shown. Scale bars, 200 μm, as indicated. (O) N27 cells were treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h and detected by CellROX Green using flow cytometry. Representative histograms for fluorescence of CellROX Green and the average fluorescence are shown, n = 6 wells from one representative of two independent experiments. (P) The representative images of H 2 DCFDA staining in N27 cells treated with DHA (1.5 μM), RSL-3 (100 nM), and NAC (1 mM) for 12 h, and the average fluorescence intensity are shown. Scale bars, 200 μm, as indicated. (Q) Cell viability of N27 cells treated with NAC and DHA (1.5 μM) and RSL-3 (100 nM) for 48 h, n = 6 wells from one representative of two independent experiments. (R) The representative images of H 2 DCFDA staining in N27 cells treated with DHA (1.5 μM), RSL-3 (100 nM), and DFO (50 μM) for 12 h, and the average fluorescence intensity are shown. Scale bars, 200 μm, as indicated. Data are means ± SEM, n = 3 wells from one representative of two independent experiments unless specified. One-way ANOVA was performed.

Article Snippet: Alpha Tubulin Monoclonal antibody (1:10000) , Proteintech , 66031-1-Ig; RRID: AB_11042766.

Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, 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: 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: Histone H4K5la activates transcription of HDAC4 in FSH-treated GCs. (A) Elaborate examination of H4K5la binding at various genomic locations within target genes. (B) Strategy for identifying specific downstream targets of H4K5la based on CUT&Tag data. (C) Computational biology suggests that HDAC4 is a possible H4K5la effector. (D) Representative Integrative Genomics Viewer (IGV) tracks showing enriched H4K5la modifications at the HDAC4 promoter in mGCs using CUT&Tag analysis. (E) Assessment of HDAC4 mRNA abundance via qRT-PCR in mGCs and KGN cells exposed to 15 mM oxamate for 2 h, and then subjected to 5 IU of FSH for 12 h. (F) qRT-PCR was performed to assess HDAC4 mRNA expression in mGCs and KGN cells after 12-h transfection with LDHA- and LDHB-targeting siRNAs, followed by a 12-h exposure to 5 IU of FSH. (G) ChIP-qPCR revealed H4K5la enrichment at the HDAC4 promoter in KGN cells pretreated with 15 mM oxamate for 2 h before FSH stimulation (5 IU, 12 h). (H) ChIP-qPCR demonstrated H4K5la binding to the HDAC4 promoter in KGN cells transfected with LDHA and LDHB siRNAs for 12 h, followed by 5 IU of FSH treatment for an additional 12 h. (I) ChIP-qPCR analysis indicated H4K5la occupancy at the HDAC4 promoter in KGN cells incubated with 10 μM C646 for 2 h prior to 12-h FSH (5 IU) exposure. (J) Western blotting was used to evaluate HDAC4 protein levels in GCs treated with 15 mM oxamate for 2 h, followed by 5 IU of FSH for 12 h. (K) HDAC4 protein expression in (J) was quantified by densitometry and normalized to TUBA1A as a loading control. (L) Western blot detection of HDAC4 expression in mGCs and KGN cells after 12 h siRNA knockdown of LDHA/LDHB and subsequent 12-h 5-IU FSH exposure. (M) The protein levels of HDAC4 in (L) were quantitatively analyzed with normalization to TUBA1A. (N) Immunoblotting analysis assessing HDAC4 protein abundance in mGCs and KGN cells after 2-h exposure to 10 μM C646, and then 12 h of 5-IU FSH administration. (O) The protein levels of HDAC4 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: Binding Assay, Quantitative RT-PCR, Expressing, Transfection, ChIP-qPCR, Incubation, Western Blot, Control, Knockdown, Quantitative Proteomics

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: Histone lactylation promotes mitochondrial biogenesis in GCs via HDAC4. (A) Western blot analysis of HDAC4 protein levels in KGN cells cultured with different concentrations of LMK-235 for 12 h. (B) The protein levels of HDAC4 in (A) were quantitatively analyzed with normalization to TUBA1A. (C) qRT-PCR quantification of mitochondrial DNA ( MT-CO2 and D-Loop ) in KGN cells after 2-h exposure to 15 mM LMK-235, and then 12 h with 5 IU of FSH. β-Actin served as the loading control for data normalization. (D) TOM20 protein levels in mGCs and KGN cells were analyzed by Western blot under the following treatment: pretreatment with 15 mM LMK-235 for 2 h, followed by stimulation with 5 IU of FSH for 12 h. (E) TOM20 protein levels in (D) were quantified and normalized to TUBA1A. (F) mGCs and KGN were pretreated with 15 mM LMK-235 for 2 h and then exposed to 5 IU of FSH for 12 h. Mitochondrial labeling was performed using MitoTracker Green (Mito Green) (green), and samples were visualized via confocal microscopy. Scale bar, 5 μm. (G) Quantitative analysis of MitoTracker Green fluorescence intensity from (F). (H) Western blot examination of HDAC4 protein abundance in KGN cells treated with HDAC4 siRNA or control siRNA over a 24-h period. (I) The protein levels of HDAC4 in (H) were quantitatively analyzed with normalization to TUBA1A. (J) qRT-PCR evaluation of mitochondrial DNA copy number in KGN cells post-HDAC4 siRNA transfection for 12 h, and then treated with 5 IU of FSH for another 12 h. β-Actin served as the loading control for data normalization. (K) Western blot assessment of TOM20 expression in KGN cells after HDAC4 siRNA transfection (12 h) and subsequent FSH treatment (5 IU, 12 h). (L) The protein levels of TOM20 in (K) were quantitatively analyzed with normalization to TUBA1A. (M) KGN cells received 12 h of HDAC4-specific siRNA transfection and then underwent 12 h of 5-IU FSH treatment. Mitochondria were visualized using MitoTracker Green and imaged by laser confocal scanning microscopy. Scale bar, 5 μm. (N) Quantitative analysis of MitoTracker Green fluorescence intensity from (M). (O) KGN cells received HDAC4-targeting siRNAs for 12 h and then 5 IU of FSH for 12 h. Subsequently, OCR was quantified. (P) KGN cells underwent HDAC4 siRNA transfection for 12 h and then received 5 IU of FSH for another 12 h. JC-1 staining measured mitochondrial membrane potential. (Q) The membrane potential levels in (P) were analyzed. 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, Cell Culture, Quantitative RT-PCR, Control, Labeling, Confocal Microscopy, Fluorescence, Quantitative Proteomics, Transfection, Expressing, Confocal Laser Scanning Microscopy, Staining, Membrane

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