Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: Expression of the KDM4A protein after lentiviral infection. (A) qPCR results of the knockdown of the protein. Expression data are normalized to the Ctrl (b-actin, transcript level). Each experiment was performed in triplicate. Bars under the same symbol (*) are statistically different under the two-tailed, unpaired Student´s t-test compared to the Ctrl expression level. *p < 0.05. (B) KDM4A protein knockdown during the differentiation protocol. Proteins were extracted at the end of each differentiation stage and detected using specific antibodies against KDM4A. HepG2 and iPSCs WT (wild type) were used as controls. S0-KD, Stage 0 knockdown; S1-KD, Stage 1 knockdown; S2-KD, Stage 2 knockdown; S3-KD, Stage 3 knockdown; S4-KD, Stage 4 knockdown; KDM4A-KD, Histone lysine demethylase 4A knockdown.

Article Snippet: Primary antibodies against forkhead box A2 (FOXA2) (710730), SRY-box transcription factor 17 (SOX17) (PA5-72815), hepatocyte nuclear factor alpha (HNF4A) (PA5-82159), SRY-box transcription factor 9 (SOX9) (PA5-81966), pancreas associated transcription factor 1a (PTF1A) (PA5-112677), and Neurogenin 3 (NeuroG3) (703206),c and goat anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibody ( G21234 ), Luria Broth Base (Miller ́s LB Broth Base) (1295027), PureLinkTM HiPure MaxiPrep kit (K210006), and Fluoromount-GTM, with DAPI (00-4959-52) were obtained from Invitrogen, United States; Recombinant pancreatic and duodenal homeobox 1 (PDX1) (ab219207), and anti-NKX6.1 antibody (ab221549) were obtained from Abcam, United States; CoraLite ® Plus 488-conjugated INS Monoclonal antibody (CL488-66198), and KDM4A polyclonal antibody (29977-1-AP) were acquired from Proteintech, United States; QuickExtractTM RNA extraction kit (QER090150) was obtained from Biosearch Technologies, United States, and OneScript ® Plus Reverse Transcriptase (G237) was obtained from ABN (Canada).

Techniques: Expressing, Infection, Knockdown, Two Tailed Test

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: Schematic representation of the differentiation process of the iPSCs KDM4A knockdown (iPSCs-KD) to pancreatic β-like cells (PβLCs). iPSCs were cultured in a 12-well plate coated with vitronectin XF™ in a sequential protocol for 13 days. The mTeSR1™ was supplemented with different cofactors and small molecules. Images were captured with a phase contrast microscope (ZEISS AX10). DE, Definitive endoderm; PG, Pancreatic gut tube; PP, Pancreatic progenitor; EP, Endocrine progenitor; PβLC, Pancreatic beta-like cell; d, days. Black bar 50 µm.

Article Snippet: Primary antibodies against forkhead box A2 (FOXA2) (710730), SRY-box transcription factor 17 (SOX17) (PA5-72815), hepatocyte nuclear factor alpha (HNF4A) (PA5-82159), SRY-box transcription factor 9 (SOX9) (PA5-81966), pancreas associated transcription factor 1a (PTF1A) (PA5-112677), and Neurogenin 3 (NeuroG3) (703206),c and goat anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibody ( G21234 ), Luria Broth Base (Miller ́s LB Broth Base) (1295027), PureLinkTM HiPure MaxiPrep kit (K210006), and Fluoromount-GTM, with DAPI (00-4959-52) were obtained from Invitrogen, United States; Recombinant pancreatic and duodenal homeobox 1 (PDX1) (ab219207), and anti-NKX6.1 antibody (ab221549) were obtained from Abcam, United States; CoraLite ® Plus 488-conjugated INS Monoclonal antibody (CL488-66198), and KDM4A polyclonal antibody (29977-1-AP) were acquired from Proteintech, United States; QuickExtractTM RNA extraction kit (QER090150) was obtained from Biosearch Technologies, United States, and OneScript ® Plus Reverse Transcriptase (G237) was obtained from ABN (Canada).

Techniques: Knockdown, Cell Culture, Microscopy

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: Western blot result of the differentiation process. The figure represents all the proteins detected during different differentiation stages of the iPSCs KDM4A-KD. Proteins are detected individually at each differentiation stage. A HepG2 cell line was used as the control for the experiment. The approximate molecular weight of the protein (kDa) is shown. SOX17–44 kDa; FOXA2–49 kDa; HNF4A 52 kDa; PDX1–40 kDa; SOX9–63 kDa; PTF1A 42 kDa; NKX6.1–50 kDa. S0, Stage 0; S1, Stage 1; S2, Stage 2; S3, Stage 3; S4, Stage 4.

Article Snippet: Primary antibodies against forkhead box A2 (FOXA2) (710730), SRY-box transcription factor 17 (SOX17) (PA5-72815), hepatocyte nuclear factor alpha (HNF4A) (PA5-82159), SRY-box transcription factor 9 (SOX9) (PA5-81966), pancreas associated transcription factor 1a (PTF1A) (PA5-112677), and Neurogenin 3 (NeuroG3) (703206),c and goat anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibody ( G21234 ), Luria Broth Base (Miller ́s LB Broth Base) (1295027), PureLinkTM HiPure MaxiPrep kit (K210006), and Fluoromount-GTM, with DAPI (00-4959-52) were obtained from Invitrogen, United States; Recombinant pancreatic and duodenal homeobox 1 (PDX1) (ab219207), and anti-NKX6.1 antibody (ab221549) were obtained from Abcam, United States; CoraLite ® Plus 488-conjugated INS Monoclonal antibody (CL488-66198), and KDM4A polyclonal antibody (29977-1-AP) were acquired from Proteintech, United States; QuickExtractTM RNA extraction kit (QER090150) was obtained from Biosearch Technologies, United States, and OneScript ® Plus Reverse Transcriptase (G237) was obtained from ABN (Canada).

Techniques: Western Blot, Control, Molecular Weight

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: Gene expression during differentiation. Line bars represent gene expression in WT iPSCs throughout the differentiation protocol, while dotted bars represent gene expression in KDM4A-KD iPSCs. Expression levels were normalized to the control (β-actin transcript). Each experiment was performed in triplicate. Bars marked with the same symbol (*) indicate a statistically significant difference compared to the control group (two-tailed, unpaired Student’s t-test, *p < 0.05). Bars marked with the same symbol (**) indicate a statistically significant difference between the two groups (WT and KDM4A-KD, two-tailed, unpaired Student’s t-test, **p < 0.05). (A) Stage 0 (DE); (B) Stage 1 Gene expression (PG); (C) Stage 2 Gene expression (PP); (D) Stage 3 Gene expression (EP); (E) Stage 4 Gene expression (PβLC). foxA2, Forkhead box A2; sox17, SRY-box transcription factor 17; hnf4A, Hepatocyte nuclear factor 4 alpha; pdx1, Pancreatic and duodenal homeobox 1; sox9, SRY-box transcription factor 9; nkx6.1, Nk6 homeobox 1 protein; ngn 3, Neurogenin-3; ins, Insulin; ptf1A, Pancreas associated transcription factor 1A; sst, Somatostatin; cgc, Glucagon.

Article Snippet: Primary antibodies against forkhead box A2 (FOXA2) (710730), SRY-box transcription factor 17 (SOX17) (PA5-72815), hepatocyte nuclear factor alpha (HNF4A) (PA5-82159), SRY-box transcription factor 9 (SOX9) (PA5-81966), pancreas associated transcription factor 1a (PTF1A) (PA5-112677), and Neurogenin 3 (NeuroG3) (703206),c and goat anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibody ( G21234 ), Luria Broth Base (Miller ́s LB Broth Base) (1295027), PureLinkTM HiPure MaxiPrep kit (K210006), and Fluoromount-GTM, with DAPI (00-4959-52) were obtained from Invitrogen, United States; Recombinant pancreatic and duodenal homeobox 1 (PDX1) (ab219207), and anti-NKX6.1 antibody (ab221549) were obtained from Abcam, United States; CoraLite ® Plus 488-conjugated INS Monoclonal antibody (CL488-66198), and KDM4A polyclonal antibody (29977-1-AP) were acquired from Proteintech, United States; QuickExtractTM RNA extraction kit (QER090150) was obtained from Biosearch Technologies, United States, and OneScript ® Plus Reverse Transcriptase (G237) was obtained from ABN (Canada).

Techniques: Gene Expression, Expressing, Control, Two Tailed Test

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: ELISA measurements of human insulin. Basal insulin was measured one-hour post-treatment with 2 mM glucose (close Barr). Stimulated insulin was measured one-hour post-treatment with 20 mM glucose (open Barr). All measurements were performed by triplicate. PβLC-KD, pancreatic β-like cells KDM4A-KD; PβLC, pancreatic β-like cells; Insulin-secreting cells (INS823/13); iPSC WT, Induced pluripotent cells without differentiation; HEK293T, Human embryonic kidney cells.

Article Snippet: Primary antibodies against forkhead box A2 (FOXA2) (710730), SRY-box transcription factor 17 (SOX17) (PA5-72815), hepatocyte nuclear factor alpha (HNF4A) (PA5-82159), SRY-box transcription factor 9 (SOX9) (PA5-81966), pancreas associated transcription factor 1a (PTF1A) (PA5-112677), and Neurogenin 3 (NeuroG3) (703206),c and goat anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibody ( G21234 ), Luria Broth Base (Miller ́s LB Broth Base) (1295027), PureLinkTM HiPure MaxiPrep kit (K210006), and Fluoromount-GTM, with DAPI (00-4959-52) were obtained from Invitrogen, United States; Recombinant pancreatic and duodenal homeobox 1 (PDX1) (ab219207), and anti-NKX6.1 antibody (ab221549) were obtained from Abcam, United States; CoraLite ® Plus 488-conjugated INS Monoclonal antibody (CL488-66198), and KDM4A polyclonal antibody (29977-1-AP) were acquired from Proteintech, United States; QuickExtractTM RNA extraction kit (QER090150) was obtained from Biosearch Technologies, United States, and OneScript ® Plus Reverse Transcriptase (G237) was obtained from ABN (Canada).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: (A) Immunofluorescence staining showing insulin (green), DAPI (blue), and merge pictures. The results showed that PβLC and PβLC-KD express insulin in response to glucose. However, insulin production in response to glucose by PβLC-KD was lower than that of PβLC, which would be consistent with the results obtained by qRT-PCR. Scale bar = 50 µm. All images were obtained on an Eclipse Ni-E microscope (Nikon) and analyzed with ImageJ software (National Institutes of Health) at 80 ms of exposure. (B) Insulin expression percentage. The fluorescence expressed by insulin was normalized to that expressed by the nucleus. Bars under the same symbol (*) are statistically different under the two-tailed, unpaired Student’s t-test (p < 0.05*; p < 0.05** n = 3). A. PβLC: Pancreatic β-like cells; B. PβLC-KD: Pancreatic β-like cells KDM4A-KD. C. HepG2: Hepatoblastoma cell line (negative control). D. INS823/13: Rat insulinoma cell line (insulin-secreting cells).

Article Snippet: Primary antibodies against forkhead box A2 (FOXA2) (710730), SRY-box transcription factor 17 (SOX17) (PA5-72815), hepatocyte nuclear factor alpha (HNF4A) (PA5-82159), SRY-box transcription factor 9 (SOX9) (PA5-81966), pancreas associated transcription factor 1a (PTF1A) (PA5-112677), and Neurogenin 3 (NeuroG3) (703206),c and goat anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibody ( G21234 ), Luria Broth Base (Miller ́s LB Broth Base) (1295027), PureLinkTM HiPure MaxiPrep kit (K210006), and Fluoromount-GTM, with DAPI (00-4959-52) were obtained from Invitrogen, United States; Recombinant pancreatic and duodenal homeobox 1 (PDX1) (ab219207), and anti-NKX6.1 antibody (ab221549) were obtained from Abcam, United States; CoraLite ® Plus 488-conjugated INS Monoclonal antibody (CL488-66198), and KDM4A polyclonal antibody (29977-1-AP) were acquired from Proteintech, United States; QuickExtractTM RNA extraction kit (QER090150) was obtained from Biosearch Technologies, United States, and OneScript ® Plus Reverse Transcriptase (G237) was obtained from ABN (Canada).

Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Microscopy, Software, Expressing, Fluorescence, Two Tailed Test, Negative Control

Journal: Clinical and Molecular Hepatology

Article Title: Glutamate dehydrogenase 1-dependent α-ketoglutarate promotes hepatitis B virus transcription by modulating histone methylations on the covalently closed circular DNA minichromosome

doi: 10.3350/cmh.2024.0694

Figure Lengend Snippet: GDH1-derived αKG interacts with KDM4A and downregulates histone methylation of H3K4, H3K9 and H4K20 on cccDNA minichromosome. (A) HBV-infected HepG2-NTCP cells were transduced with lentivirus-expressed shGDH1 for 5 days to measure KDMs demethylase activity. (B) HBV-infected cells transduced with GDH1-expressing lentivirus subjected to detect KDM4A demethylase activity. (C, D) The interaction between KDM4A and GDH1 was detected by immunoprecipitation and PLA in HepG2-NTCP cells. (E, F) MST analysis was performed by using Monolith NT.115 (NanoTemper Technologies). MST traces for single dose of αKG (right panel) and dose response curve (left panel) for KDM4A WT (E) and KDM4A mut (F) were provided. (G) ChIP assay revealed the enrichment of KDM4A, KDM1A, KDM1B on cccDNA, GAPDH , MYH6 promoter. (H) HBV-infected cells transduced with GDH1 or treated with DM-αKG, ChIP assay showed the enrichment of H3K4me3, H3K9me3, H4K20me3 on cccDNA, GAPDH , MYH6 promoters. (I) Localization of H3K4me3, H3K9me3, H4K20me3 along the HBV genome was exhibited by using ChIP-Seq. ** P <0.01; *** P <0.001. GDH1, glutamate dehydrogenase 1; αKG, α-ketoglutarate; cccDNA, covalently closed circular DNA; HBV, hepatitis B virus; KDMs, histone lysine demethylases; PLA, proximity ligation assay; MST, micro scale thermophoresis; WT, wild-type; ChIP, chromatin immunoprecipitation; DM-αKG, dimethyl-αKG; LSD, lysine-specific histone demethylase.

Article Snippet: Assay was performed by using JMJD2A (KDM4A) Homogenous Assay Kit (#50413; BPS Bioscience, Milan, Italy) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Methylation, Infection, Transduction, Activity Assay, Expressing, Immunoprecipitation, ChIP-sequencing, Virus, Proximity Ligation Assay, Chromatin Immunoprecipitation