Journal: Nucleic Acids Research

Article Title: Lactylation of CREB is required for FSH-induced proliferation and differentiation of ovarian granulosa cells

doi: 10.1093/nar/gkaf882

Figure Lengend Snippet: FSH promotes CREB lactylation in GCs. ( A ) Each mouse was intraperitoneally injected with 10, 5, 2, and 2 IU FSH (dissolved in 0.9% saline) every 12 h and control mice were concomitantly injected with the same volume of 0.9% saline. After 48 h, mice in each group were sacrificed for the collection of GCs to identify lactylation by LC-MS/MS. ( B ) Enumeration of proteins with increased lactylation. ( C ) Heatmap showing profiles of all quantifiable Kla sites before or after normalization according to quantitative proteomics analysis. Before normalization, lactylation levels were quantified without proteomic normalization; after normalization, lactylation levels were quantified with proteomic normalization. ( D ) Gene Ontology analysis of the proteins showing increased lactylation. ( E ) Subcellular classification of the proteins exhibiting increased lactylation. ( F ) Validation of the conservation of the CREB S133 phosphorylation site and S136 lactylation site across different species. ( G ) Co-immunoprecipitation assays confirmed the interaction between CREB and Pan-Kla in KGN cells following treatment with 5 IU of FSH for 12 h. ( H ) The ratio of lactylated CREB to total CREB was quantified in (G) using densitometric analysis. ( I ) Co-immunoprecipitation revealed CREB–Pan-Kla interaction in KGN cells treated with 15 mM oxamate for 2 h followed by stimulation with 5 IU of FSH for 12 h. ( J ) The ratio of lactylated CREB to total CREB was quantified in (I) using densitometric analysis. ( K ) Co-immunoprecipitation detection of CREB interaction with Pan-Kla after simultaneous knockdown of LDHA and LDHB for 12 h and then treatment with 5 IU of FSH in KGN cells. ( L ) The ratio of lactylated CREB to total CREB was quantified in (K) using densitometric analysis. ( M ) Co-immunoprecipitation detection of CREB interaction with Pan-Kla after transfection with ACSS2 and SUCLG1 siRNAs for 12 h and then treatment with 5 IU of FSH in KGN cells. ( N ) The ratio of lactylated CREB to total CREB was quantified in (M) using densitometric analysis. ( O ) The 3D structure of the K136R mutant CREB. ( P ) Co-immunoprecipitation detection of CREB interaction with Pan-Kla after overexpression of Flag-tagged WT or K136R CREB in KGN cells. ( Q ) The ratio of lactylated CREB to total CREB was quantified in (P) using densitometric analysis. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. NS indicates no difference.

Article Snippet: Antibodies against p-CREB (28792-1-AP), CyclinD2 (10934-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), Ki67 (28074-1-AP), Pan-Acetylation (66289-1-Ig), AARS1 (67909-1-Ig), AARS1 (22696-1-Ig), TIP60/KAT5 (10827-1-Ig), SUCLG1 (14923-1-Ig), ACSS2 (16087-1-Ig), and LDHA (21799-1-AP) were purchased from Proteintech.

Techniques: Injection, Saline, Control, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Biomarker Discovery, Phospho-proteomics, Immunoprecipitation, Knockdown, Transfection, Mutagenesis, Over Expression

Journal: Nucleic Acids Research

Article Title: Lactylation of CREB is required for FSH-induced proliferation and differentiation of ovarian granulosa cells

doi: 10.1093/nar/gkaf882

Figure Lengend Snippet: Suppression of P300 activity inhibits FSH-induced GCs proliferation. ( A ) Co-immunoprecipitation analysis demonstrated a marked attenuation of CREB binding to Pan-Kla after knockdown of lactyltransferases for 24 h in KGN cells. ( B ) Western blot analysis was performed to evaluate Pan-Kla levels in mGCs and KGN cells pre-treated with 10 μM C646 for 2 h prior to stimulation with 5 IU of FSH for 12 h. ( C ) Co-immunoprecipitation analysis demonstrated a marked attenuation of CREB binding to Pan-Kla pre-treated with 10 μM C646 for 2 h prior to stimulation with 5 IU of FSH for 12 h in KGN. ( D ) Quantitative analysis of the lactylated CREB proportion relative to total CREB was performed in (C). ( E ) Western blot analysis was performed to evaluate Pan-Kla levels in KGN cells pre-treated with 10 μM C646 for 2 h prior to 15 mM sodium lactate stimulation for 12 h. ( F ) Co-immunoprecipitation analysis demonstrated a marked attenuation of CREB binding to Pan-Kla pre-treated with 10 μM C646 for 2 h prior to 15 mM sodium lactate stimulation for 12 h in KGN. ( G ) Quantitative analysis of the lactylated CREB proportion relative to total CREB was performed in (F). ( H ) Western blot assessing acetylation protein expression in KGN cells treated with 5 IU of FSH. ( I ) Western blot assessing acetylation protein expression after KGN treatment with 10 μM C646 for 2 h followed by treatment with 5 IU of FSH for 12 h. ( J ) Western blot assessing acetylation protein expression in different cells after treatment with C646 for 12h. ( K ) Cell viability was assessed using the CCK-8 assay following treatment with 10 μM C646 followed by 5 IU of FSH in mGCs and KGN cells. ( L ) Western blot assessing levels of proliferation-related proteins in mGCs and KGN cells treated with 5 IU of FSH and/or 10 μM C646. ( M ) Cell viability in mGCs and KGN cells was quantitatively evaluated using the CCK-8 assay following sequential transfection with P300 siRNA for 12 h and subsequent stimulation with 5 IU of FSHfor 12 h. ( N ) Western blot analysis was performed to evaluate expression of proliferation-related proteins in mGCs and KGN cells following sequential treatments: transfected with P300 siRNA for 12 h, followed by FSH stimulation for 12 h. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. NS indicates no difference.

Article Snippet: Antibodies against p-CREB (28792-1-AP), CyclinD2 (10934-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), Ki67 (28074-1-AP), Pan-Acetylation (66289-1-Ig), AARS1 (67909-1-Ig), AARS1 (22696-1-Ig), TIP60/KAT5 (10827-1-Ig), SUCLG1 (14923-1-Ig), ACSS2 (16087-1-Ig), and LDHA (21799-1-AP) were purchased from Proteintech.

Techniques: Activity Assay, Immunoprecipitation, Binding Assay, Knockdown, Western Blot, Expressing, CCK-8 Assay, Transfection

Journal: Nucleic Acids Research

Article Title: Lactylation of CREB is required for FSH-induced proliferation and differentiation of ovarian granulosa cells

doi: 10.1093/nar/gkaf882

Figure Lengend Snippet: FSH-induced CREB lactylation facilitates CREB phosphorylation. ( A ) Schematic representation of the three-dimensional structure of CREB. ( B ) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 15 mM 2-DG for 2 h followed by treatment with 5 IU of FSH for 12 h. ( C ) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 15 mM oxamate for 2 h followed by treatment with 5 IU of FSH for 12 h. ( D ) Western blot detecting P-CREB (S133) protein levels after transfection with LDHA and LDHB siRNAs for 12 h and subsequent stimulation with 5 IU of FSH for 12 h. ( E ) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 3 mM α-CHCA for 2 h followed by 15 mM sodium lactate treatment for 12 h. ( F ) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 10 μM C646 for 2 h followed by treatment with 5 IU of FSH for 12 h. ( G ) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 10 μM C646 for 2 h followed by 15 mM sodium lactate treatment for 12 h. ( H ) Western blot detecting P-CREB (S133) protein levels after overexpression of WT-Flag-CREB or MUT-Flag-CREB K136R in CREB knockdown KGN cells with FSH treatment. ( I ) CREB expression levels were compared between WT KGN cells and CREB knockout (KO) KGN cells. ( J ) Western blot detecting P-CREB (S133) protein levels after overexpression of WT-Flag-CREB or MUT-Flag-CREB K136R with treatment with 10 IU of FSH in CREB KO KGN cells. ( K ) A schematic diagram illustrating the lactylated CREB–PKA kinase reaction system. ( L ) Detection of the phosphorylation or lactylation level in the Flag-tagged CREB–bead complexes. ( M ) The levels of phosphorylated CREB and lactylated CREB in (L) were quantified using densitometric analysis. ( N ) Molecular docking analysis of WT CREB and the K136R mutant CREB with the PRKACA protein. Binding affinity was assessed based on the PIPER pose energy docking score. ( O ) Co-immunoprecipitation detection of CREB interaction with PRKACA after overexpression of WT-Flag-CREB or MUT-Flag-CREB K136R with treatment with 5 IU of FSH in KGN cells. ( P ) Co-immunoprecipitation assays were performed to assess the interaction between CREB and Pan-Kla in KGN cells overexpressing Flag-tagged WT CREB or its phospho-deficient S133A mutant in KGN cells treated with 5 IU of FSH. ( Q ) Co-immunoprecipitation assays were conducted to compare the physical interaction between CREB and P300 in KGN cells following transient transfection with Flag-tagged WT CREB or its phospho-deficient S133A mutant in KGN cells treated with 5 IU of FSH. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. NS indicates no difference.

Article Snippet: Antibodies against p-CREB (28792-1-AP), CyclinD2 (10934-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), Ki67 (28074-1-AP), Pan-Acetylation (66289-1-Ig), AARS1 (67909-1-Ig), AARS1 (22696-1-Ig), TIP60/KAT5 (10827-1-Ig), SUCLG1 (14923-1-Ig), ACSS2 (16087-1-Ig), and LDHA (21799-1-AP) were purchased from Proteintech.

Techniques: Phospho-proteomics, Western Blot, Transfection, Over Expression, Knockdown, Expressing, Knock-Out, Mutagenesis, Protein Binding, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Lactylation of CREB is required for FSH-induced proliferation and differentiation of ovarian granulosa cells

doi: 10.1093/nar/gkaf882

Figure Lengend Snippet: CREB lactylation promotes GCs proliferation by activating CREB-dependent transcription. ( A ) qRT–PCR was performed to measure the expression levels of Cyclin D2 and c-FOS mRNAs in CREB KO KGN cells after overexpression of WT-CREB or MUT-Flag-CREB K136R for 12 h followed by treatment with 5 IU of FSH for 12 h. ( B ) ChIP assays were performed to evaluate the recruitment of Flag-tagged CREB to Cyclin D2 and c-FOS promoter regions in CREB KO KGN cells after overexpression of WT-CREB or MUT-CREB K136R for 12 h followed by treatment with 5 IU of FSH for 12 h. ( C ) Cell viability was assessed in FSH (5 IU)-treated CREB KO KGN cells following introduction of the WT-CREB or lysine-deficient K136R mutation (MUT-CREB K136R ). ( D ) Western blot detecting proliferation-related protein levels after transfection with WT-Flag-CREB or WT-CREB K136R for 12 h followed by treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. ( E ) CREB KO KGN cells underwent WT-CREB transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. Cell viability was assessed using the CCK-8 assay. ( F ) CREB KO KGN cells underwent WT-CREB transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. The proliferation-related protein levels were assessed by western blot. ( G ) CREB KO KGN cells underwent MUT-CREB S133D transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. Cell viability was assessed using the CCK-8 assay. ( H ) CREB KO KGN cells underwent MUT-CREB S133D transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. The proliferation-related protein levels were assessed by western blot. ( I ) CREB KO KGN cells underwent WT-CREB/MUT-CREB S133A transfection for 12 h or treatement with 50 mM H-89 for 2 h prior to treatment with 5 IU of FSH for 12 h. Cell viability was assessed using the CCK-8 assay. ( J ) CREB KO KGN cells underwent WT-CREB/MUT-CREB S133A transfection for 12 h or treatment with 50 mM H-89 for 2 h prior to treatment with 5 IU of FSH for 12 h. The proliferation-related protein levels were assessed by western blot. ( K ) CREB KO KGN cells underwent S133WT-S136WT, S133D-K136WT, and S133D-K136R transfection for 12 h prior to treatment with 5 IU of FSH for 12 h. Cell viability was assessed using the CCK-8 assay. ( L ) CREB KO KGN cells underwent S133WT-S136WT, S133D-K136WT, and S133D-K136R transfection for 12 h prior to treatment with 5 IU of FSH for 12 h. Western blot analysis of proliferation-related protein levels after the indicated treatments. ( M ) Cyclin D2 promoter activity was examined by a dual-luciferase reporter gene assay after overexpression of WT-CREB or MUT-Flag-CREB K136R for 12 h followed by treatment with 5 IU of FSH for 12 h in CREB KO cells. ( N ) A dual-luciferase reporter gene assay of Cyclin D2 promoter activity was assessed in FSH (5 IU)-treated CREB KO KGN cells following introduction of the WT-CREB. ( O ) Detection of Cyclin D2 promoter activity using dual-luciferase assay after CREB KO KGN cells underwent MUT-CREB S133D transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. ( P ) The dual-luciferase reporter assay system was employed to quantitatively assess transcriptional activity of the Cyclin D2 promoter after KGN cells underwent WT-CREB/MUT-CREB S133A transfection for 12 h or treatment with 50 mM H-89 for 2 h prior to treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. ( Q ) The transcriptional activity of the Cyclin D2 promoter was quantitatively evaluated using the dual-luciferase reporter assay system after KGN cells underwent S133WT-S136WT, S133D-K136WT, and S133D-K136R transfection for 12 h prior to treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. ( R ) Comparative co-immunoprecipitation analysis was performed to characterize the interaction between CBP/P300 and Flag-tagged CREB variants in FSH-treated KGN cells. Specifically, cells were transiently transfected with either WT Flag-CREB or the lactylation-defective Flag-CREB K136R mutant prior to 12 h FSH stimulation. ( S ) Co-immunoprecipitation detection of CREB interaction with CBP/P300 after transfection with Flag-CREB/Flag-CREB S133A for 12 h, or treatment with 50 nM H-89 prior to treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. ( T ) Co-immunoprecipitation detection of CREB interaction with CBP/P300 after transfection with S133WT-S136WT, S133D-K136WT, and S133D-K136R for 12 h prior to treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. NS indicates no difference.

Article Snippet: Antibodies against p-CREB (28792-1-AP), CyclinD2 (10934-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), Ki67 (28074-1-AP), Pan-Acetylation (66289-1-Ig), AARS1 (67909-1-Ig), AARS1 (22696-1-Ig), TIP60/KAT5 (10827-1-Ig), SUCLG1 (14923-1-Ig), ACSS2 (16087-1-Ig), and LDHA (21799-1-AP) were purchased from Proteintech.

Techniques: Quantitative RT-PCR, Expressing, Over Expression, Mutagenesis, Western Blot, Transfection, CCK-8 Assay, Activity Assay, Luciferase, Reporter Gene Assay, Reporter Assay, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Lactylation of CREB is required for FSH-induced proliferation and differentiation of ovarian granulosa cells

doi: 10.1093/nar/gkaf882

Figure Lengend Snippet: CREB lactylation promotes GCs differentiation by activating CREB-dependent transcription. ( A ) Treatment of mGCs with 15 mM 2-DG or 15 mM oxamate for 2 h followed by 5 IU of FSH for 12 h. The levels of estradiol (E2) in the culture medium were examined by radioimmunoassay. ( B ) mGC treatment with 15 mM 2-DG or 15 mM oxamate for 2 h followed by 5 IU of FSH for 12 h. The levels of progesterone (P4) in the culture medium were examined by radioimmunoassay. ( C ) Western blot determining protein levels of 3β-HSD, CYP19A1, and CYP11A1 in ovarian GCs treated with 15 mM 2-DG for 2 h followed by 5 IU of FSH for 12 h. ( D ) Western blot analysis of 3β-HSD, CYP19A1, and CYP11A1 protein expression levels in ovarian GCs treated with 15 mM oxamate for 2 h followed by stimulation with 5 IU FSH for 12 h. ( E ) mGCs transfected with LDHA and LDHB siRNAs for 12 h followed or not with 5 IU of FSH for 12 h. The culture medium was collected to examine the estradiol level using radioimmunoassay. ( F ) The levels of progesterone in the culture medium were examined by radioimmunoassay after mGCs were transfected with LDHA and LDHB siRNAs for 12 h followed or not with 5 IU of FSH for 12 h. ( G ) Western blot determining protein levels of 3β-HSD, CYP19A1, and CYP11A1 in ovarian GCs after transfection with LDHA and LDHB siRNAs for 12 h followed or not with 5 IU of FSH for 12 h. ( H ) mGCs treated with 10 μM C646 for 2 h prior to 5 IU of FSH for 12 h. The culture medium was collected for to examine the estradiol level using radioimmunoassay. ( I ) mGCs treated with 10 μM C646 for 2 h prior to 5 IU of FSH for 12 h. The culture medium was collected to examine the progesterone level using radioimmunoassay. ( J ) Western blot determining protein levels of 3β-HSD, CYP19A1, and CYP11A1 in ovarian GCs after treatment with 10 μM C646 for 2 h prior to 5 IU of FSH for 12 h. ( K ) The detection of estradiol levels in culture medium by radioimmunoassay after transfection with WT-CREB or MUT-CREB K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. ( L ) The detection of progesterone levels in culture medium by radioimmunoassay after transfection with WT-CREB or MUT-CREB K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. ( M ) Western blot determining protein levels of 3β-HSD, CYP19A1, and CYP11A1 in CREB KO KGN cells after transfection with WT-CREB or MUT-CREB K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. ( N ) qRT–PCR detecting the mRNA levels of Cyp19A1 and Cyp11A1 after transfection with WT-CREB or MUT-CREB K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. ( O ) The binding of Flag-tagged CREB to the Cyp19A1 and Cyp11A1 promoters in CREB KO KGN cells after transfection with WT-CREB or MUT-CREB K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. NS indicates no difference.

Article Snippet: Antibodies against p-CREB (28792-1-AP), CyclinD2 (10934-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), Ki67 (28074-1-AP), Pan-Acetylation (66289-1-Ig), AARS1 (67909-1-Ig), AARS1 (22696-1-Ig), TIP60/KAT5 (10827-1-Ig), SUCLG1 (14923-1-Ig), ACSS2 (16087-1-Ig), and LDHA (21799-1-AP) were purchased from Proteintech.

Techniques: RIA Assay, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Binding Assay

Journal: Nucleic Acids Research

Article Title: Lactylation of CREB is required for FSH-induced proliferation and differentiation of ovarian granulosa cells

doi: 10.1093/nar/gkaf882

Figure Lengend Snippet: In vivo validation of the mechanistic model through intraperitoneal injection of C646. ( A ) A schematic diagram depicts the intraperitoneal injection protocol for C646 and FSH. Briefly, each mouse received intraperitoneal injections of 10, 5, 2, or 2 IU FSH (dissolved in 0.9% saline) along with 15 mg/kg C646 (dissolved in 0.9% saline) every 12 h. Control mice were injected with an equal volume of 0.9% saline. ( B ) Immunohistochemical analysis of Pan-Kla levels was performed following the protocol described in (A). Scale bar: 100 μm. ( C ) Quantitative analysis of Pan-Kla levels in (B). ( D ) Western blot analysis was performed to detect Pan-Kla levels according to the protocol in (A). ( E ) Co-immunoprecipitation detection of CREB interaction with Pan-Kla according to the protocol in (A). ( F ) Western blot analysis of P-CREB (S133) protein levels according to the protocol in (A). ( G ) The interaction of CREB with PRKACA or CBP/P300 was analyzed by co-immunoprecipitation following the method in (A). ( H ) EdU incorporation assay detects the proliferation activity of mouse ovarian GCs following the method in (A). Scale bar: 100 μm. ( I ) Western blot analysis of proliferation-related protein levels according to the protocol in (A). ( J ) The levels of E2 in serum were examined by radioimmunoassay according to the protocol in (A). ( K ) The levels of progesterone in serum were examined by radioimmunoassay following the method in (A). ( L ) Western blot analysis of differentiation-related protein levels following the method in (A). ( M ) Measurement of ovarian size following the method in (A). ( N ) Measurement of ovarian weight following the method in ( A ). ( O ) Measurement of follicle diameter according to the protocol in (A). ( P ) Quantitative histomorphological analysis of ovarian follicles at various developmental stages was performed using H&E staining in mice according to the protocol in (A). PF, primary follicle; SF, secondary follicle; AF, antral follicle. Scale bar: 100 μm. ( Q ) Histomorphometric quantification of ovarian follicles at distinct developmental stages was performed as in (P). The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. NS indicates no difference.

Article Snippet: Antibodies against p-CREB (28792-1-AP), CyclinD2 (10934-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), Ki67 (28074-1-AP), Pan-Acetylation (66289-1-Ig), AARS1 (67909-1-Ig), AARS1 (22696-1-Ig), TIP60/KAT5 (10827-1-Ig), SUCLG1 (14923-1-Ig), ACSS2 (16087-1-Ig), and LDHA (21799-1-AP) were purchased from Proteintech.

Techniques: In Vivo, Biomarker Discovery, Injection, Saline, Control, Immunohistochemical staining, Western Blot, Immunoprecipitation, Activity Assay, RIA Assay, Staining