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

Journal: Iranian Journal of Pathology

Article Title: Designing and Development of a DNA Vaccine Based On Structural Proteins of Hepatitis C Virus

doi:

Figure Lengend Snippet: Agarose gel electrophoresis of coreE1-E2 PCR product. Lane 1, 2: a 224 bp PCR product; Lane 3: 1kb DNA size marker (Fermentas, Germany

Article Snippet: Lane 1: digested vector and core-E1-E2 fusion fragment; Lane 2: recombinant vector linearized by Bam HI; Lane M: 1kb DNA size marker (Fermentas, Germany fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 3 caption a7 Detection of core-E1-E2 mRNA in transfected and non-transfected Huh- 7.5 cells by RT-PCR analysis.

Techniques: Agarose Gel Electrophoresis, Marker

Journal: Iranian Journal of Pathology

Article Title: Designing and Development of a DNA Vaccine Based On Structural Proteins of Hepatitis C Virus

doi:

Figure Lengend Snippet: Double digestion of recombinant vector by BamHI and HindIII restriction enzymes that lead to excision of core-E1-E2 fusion gene. Lane 1: digested vector and core-E1-E2 fusion fragment; Lane 2: recombinant vector linearized by BamHI; Lane M: 1kb DNA size marker (Fermentas, Germany

Article Snippet: Lane 1: digested vector and core-E1-E2 fusion fragment; Lane 2: recombinant vector linearized by Bam HI; Lane M: 1kb DNA size marker (Fermentas, Germany fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 3 caption a7 Detection of core-E1-E2 mRNA in transfected and non-transfected Huh- 7.5 cells by RT-PCR analysis.

Techniques: Recombinant, Plasmid Preparation, Marker

Journal: Iranian Journal of Pathology

Article Title: Designing and Development of a DNA Vaccine Based On Structural Proteins of Hepatitis C Virus

doi:

Figure Lengend Snippet: Detection of core-E1-E2 mRNA in transfected and non-transfected Huh- 7.5 cells by RT-PCR analysis. RTPCR analysis using specific primers designated for N-terminal region of fragment showed negative results in non- transfected cells (lane3) and a band with a size of approximately 950bp in transfected cells with recombinant vector (lanes 1,2). Lane M: 1kb DNA size marker

Article Snippet: Lane 1: digested vector and core-E1-E2 fusion fragment; Lane 2: recombinant vector linearized by Bam HI; Lane M: 1kb DNA size marker (Fermentas, Germany fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 3 caption a7 Detection of core-E1-E2 mRNA in transfected and non-transfected Huh- 7.5 cells by RT-PCR analysis.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Recombinant, Plasmid Preparation, Marker

Journal: Nucleic Acids Research

Article Title: Life without tRNA Ile -lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA 2 Ile

doi: 10.1093/nar/gkt1009

Figure Lengend Snippet: Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

Article Snippet: For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

Techniques: Purification, Mutagenesis, Labeling, Control, Autoradiography, Clear Native PAGE, Staining, Northern Blot, Hybridization

Journal: Nucleic Acids Research

Article Title: Life without tRNA Ile -lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA 2 Ile

doi: 10.1093/nar/gkt1009

Figure Lengend Snippet: RNA sequencing of the anticodon loop of wild-type tRNA 1 and mutant tRNA 1 by MS analysis of RNase T1 fragments

Article Snippet: For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

Techniques: RNA Sequencing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Life without tRNA Ile -lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA 2 Ile

doi: 10.1093/nar/gkt1009

Figure Lengend Snippet: Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

Article Snippet: For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

Techniques: Binding Assay, Purification, Mutagenesis, Isolation, Concentration Assay