Journal: BMC Cancer

Article Title: Reduced repair of 8-hydroxyguanine in the human breast cancer cell line, HCC1937

doi: 10.1186/1471-2407-6-297

Figure Lengend Snippet: Expression of specific DNA repair proteins . Total proteins (50 μg) were separated by SDS-PAGE and the proteins transferred onto a PVDF membrane. The immunoblots were probed with antibodies against the specific proteins. Panel A represents basal levels of OGG1, NEIL1, p53 and APE1 in AG11134, MCF-7 and HCC1937 and, panel B shows RT-PCR showing the levels of OGG1 1a mRNA transcripts in AG11134, HCC1937 and MCF-7 cells. β-actin was used as a loading control.

Article Snippet: The membranes were blocked for 1 h in 5% dry milk in PBS-Tween (0.2%) and then probed with the following primary antibodies: OGG1 (Novus Biologicals, Littleton, CO), APE1 (Novus Biologicals), NEIL1 (Oncogene, San Diego, CA), p53 (Oncogene, San Diego, CA), SOD1 (Calbiochem, San Diego, CA), SOD2 (Abcam, Cambridge, MA) and catalase (Calbiochem).

Techniques: Expressing, SDS Page, Membrane, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal: BMC Cancer

Article Title: Reduced repair of 8-hydroxyguanine in the human breast cancer cell line, HCC1937

doi: 10.1186/1471-2407-6-297

Figure Lengend Snippet: Stimulation of 8-OH-Gua-specific incision activity of HCC1937 nuclear extracts by purified OGG1 . HCC1937 nuclear extracts (50 μg) were incubated with a 5' end-labeled oligonucleotide containing a single 8-OH-Gua in the presence of increasing amounts of hOGG1 for 1 h. Reactions were stopped and the products separated by electrophoresis through 20% polyacrylamide gels containing 7 M urea. The band images were analyzed by PhosphorImager and quantified using ImageQuant software. Panel A , a representative gel image showing the incision reaction products, panel B , quantified results of the incision reaction products. One-way ANOVA revealed a statistically significant difference of p < 0.002 (*) in the stimulation of activity by purified hOGG1. A unit of hOGG1 catalyzes the cleavage of 1 picomole of a duplex 32 P-labeled oligonucleotide in 1 h at 37°C at 8-OH-Gua/C base pair.

Article Snippet: The membranes were blocked for 1 h in 5% dry milk in PBS-Tween (0.2%) and then probed with the following primary antibodies: OGG1 (Novus Biologicals, Littleton, CO), APE1 (Novus Biologicals), NEIL1 (Oncogene, San Diego, CA), p53 (Oncogene, San Diego, CA), SOD1 (Calbiochem, San Diego, CA), SOD2 (Abcam, Cambridge, MA) and catalase (Calbiochem).

Techniques: Activity Assay, Purification, Incubation, Labeling, Electrophoresis, Software

Journal: BMC Cancer

Article Title: Reduced repair of 8-hydroxyguanine in the human breast cancer cell line, HCC1937

doi: 10.1186/1471-2407-6-297

Figure Lengend Snippet: Incision of 8-OH-Gua by HCC1937 nuclear extracts after transfection with pFlag-CMV-4- OGG1 . HCC1937 cells (2.5 × 10 6 ) were transfected with either pFlag-CMV-4 carrying hOGG1 gene or pFlag-CMV-4 without hOGG1 gene and nuclear extracts and whole cell extracts were prepared 48 h after transfection. Western blot analysis was performed using whole cell extracts and incision reactions performed using nuclear extracts and a duplex 5' end-labeled oligonucleotide containing a single 8-OH-Gua. Panel A , shows the expression of pFlag-CMV-4-OGG1 in HCC1937 cells and, panel B , shows the incision of 8-OH-Gua by nuclear extracts containing the transfected pFlag-CMV-4-OGG1 or just the vector alone.

Article Snippet: The membranes were blocked for 1 h in 5% dry milk in PBS-Tween (0.2%) and then probed with the following primary antibodies: OGG1 (Novus Biologicals, Littleton, CO), APE1 (Novus Biologicals), NEIL1 (Oncogene, San Diego, CA), p53 (Oncogene, San Diego, CA), SOD1 (Calbiochem, San Diego, CA), SOD2 (Abcam, Cambridge, MA) and catalase (Calbiochem).

Techniques: Transfection, Western Blot, Labeling, Expressing, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: Association between oxidative DNA damage and the expression of 8-oxoguanine DNA glycosylase 1 in lung epithelial cells of neonatal rats exposed to hyperoxia

doi: 10.3892/mmr.2015.3339

Figure Lengend Snippet: Primers used for reverse transcription quantitative polymerase chain reaction.

Article Snippet: The sections were then blocked with 5% goat serum for 1 h at room temperature and incubated with goat polyclonal anti-OGG1 primary antibody (1:50; ab115841; Abcam) overnight at 4°C.

Techniques: Reverse Transcription

Journal: Molecular Medicine Reports

Article Title: Association between oxidative DNA damage and the expression of 8-oxoguanine DNA glycosylase 1 in lung epithelial cells of neonatal rats exposed to hyperoxia

doi: 10.3892/mmr.2015.3339

Figure Lengend Snippet: Expression of OGG1 (red stain) is predominantly localized in the cytoplasm on (A) day 1 and (B) day 5 in normoxia-exposed rats and on (C) day 1 in hyperoxia-exposed rats. On days (D) 3, (E) 5 and (F) 7 in hyperoxia (magnification, ×400), the expression of OGG1 increased in the cytoplasm and the nucleus. (G and H) Western blotting revealed similar patterns of expression. Data are expressed as the mean ± standard deviation ( * P<0.05, ** P<0.01 as compared with the normoxia group). OGG1, 8-oxoguanine DNA glycosylase 1; N, normoxia; H, hyperoxia.

Article Snippet: The sections were then blocked with 5% goat serum for 1 h at room temperature and incubated with goat polyclonal anti-OGG1 primary antibody (1:50; ab115841; Abcam) overnight at 4°C.

Techniques: Expressing, Staining, Western Blot, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Association between oxidative DNA damage and the expression of 8-oxoguanine DNA glycosylase 1 in lung epithelial cells of neonatal rats exposed to hyperoxia

doi: 10.3892/mmr.2015.3339

Figure Lengend Snippet: Protein expression of OGG1 in cultured neonatal rat alveolar epithelial type II cells. (A) Western blotting and (B) densitometric quantification of the protein expression of OGG1 following different durations of hyperoxia or normoxia exposure. Data are expressed as the mean ± standard deviation ( * P<0.05; ** P<0.01 as compared with the normoxia group). OGG1, 8-oxoguanine DNA glycosylase 1.

Article Snippet: The sections were then blocked with 5% goat serum for 1 h at room temperature and incubated with goat polyclonal anti-OGG1 primary antibody (1:50; ab115841; Abcam) overnight at 4°C.

Techniques: Expressing, Cell Culture, Western Blot, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Association between oxidative DNA damage and the expression of 8-oxoguanine DNA glycosylase 1 in lung epithelial cells of neonatal rats exposed to hyperoxia

doi: 10.3892/mmr.2015.3339

Figure Lengend Snippet: mRNA expression levels of OGG1 in lung tissues and AECII cells. (A) Neonatal rat lung tissues and (B) neonatal rat AECII cells exposed to hyperoxia or normoxia. Data are expressed as the mean ± standard deviation (P>0.05 for all comparisons). AECII, alveolar epithelial type II cells; OGG1, 8-oxoguanine DNA glycosylase 1.

Article Snippet: The sections were then blocked with 5% goat serum for 1 h at room temperature and incubated with goat polyclonal anti-OGG1 primary antibody (1:50; ab115841; Abcam) overnight at 4°C.

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Metformin prevented high glucose-induced endothelial reactive oxygen species via OGG1 in an AMPKα-Lin-28 dependent pathway

doi: 10.1101/2020.11.18.388462

Figure Lengend Snippet: High glucose decreased OGG1 expression while increased endothelial reactive oxygen species levels. (A-C) OGG1 mRNA and protein levels were tested after HG exposure for indicated periods. (B) is the statistical result of (A). (D) The expression of OGG1 in the aorta of diabetic mice were assessed via Immunofluorescence. (E-F) HUVECs were digested and incubated with DHE probe (1 μM) for 30 minutes after high glucose exposure for 12 hours. The mean intensities were obtained via flow Jo software. Scar bar =25μm. *P<0.05 versus NG group (mannitol control group). Values are the means±SD with N = 3.

Article Snippet: Primary antibodies anti-OGG1 (NB100-106, Novus Biologicals), anti-Lin28 ser 200 (20607, CST), anti-Lin28 (3695, CST), anti-AMPK (5831, CST), anti-Phospho-AMPKα (50081, CST), and ß-Actin (4970, CST) were obtained commercially.

Techniques: Expressing, Immunofluorescence, Incubation, Software, Control

Journal: bioRxiv

Article Title: Metformin prevented high glucose-induced endothelial reactive oxygen species via OGG1 in an AMPKα-Lin-28 dependent pathway

doi: 10.1101/2020.11.18.388462

Figure Lengend Snippet: Metformin decreased ROS and activated OGG1 in the HUVECs. (A-B) Endothelial ROS was measured by DHE probe after high glucose exposure with metformin or not. (B) is the stastical result of (A). (C-E) OGG1 mRNA and protein levels were tested after metformin (0.5 mM) exposure for 24 hours. (E) is the statistical result of (D). *P<0.05 versus NG group (mannitol control group). #P<0.05 versus HG group. Values are the means±SD with N = 3. (F-G) HUVECs were digested and incubated with DHE probe (1 μM) for 30 minutes after silencing OGG1 or not in the presence with HG and metformin. The mean intensities were obtained via flow Jo software. Scar bar =25μm. *P<0.05 versus HG plus metformin group. Values are the means±SD with N = 3.

Article Snippet: Primary antibodies anti-OGG1 (NB100-106, Novus Biologicals), anti-Lin28 ser 200 (20607, CST), anti-Lin28 (3695, CST), anti-AMPK (5831, CST), anti-Phospho-AMPKα (50081, CST), and ß-Actin (4970, CST) were obtained commercially.

Techniques: Control, Incubation, Software

Journal: bioRxiv

Article Title: Metformin prevented high glucose-induced endothelial reactive oxygen species via OGG1 in an AMPKα-Lin-28 dependent pathway

doi: 10.1101/2020.11.18.388462

Figure Lengend Snippet: The OGG1 expression stimulated by metformin was dependent on its mRNA stability in the HUVECs. (A) HUVECs were incubated with actinomycin D (Act D, 10 μg/ml) for 0, 6, and 12 hours, and then OGG1 mRNA was measured by qPCR. (B-C) OGG1 protein levels were tested by WB after CHX-treated HUVECs for 0, 6, and 12 hours. *P<0.05 versus control group (mannitol control group). Values are the means±SD with N = 3.

Article Snippet: Primary antibodies anti-OGG1 (NB100-106, Novus Biologicals), anti-Lin28 ser 200 (20607, CST), anti-Lin28 (3695, CST), anti-AMPK (5831, CST), anti-Phospho-AMPKα (50081, CST), and ß-Actin (4970, CST) were obtained commercially.

Techniques: Expressing, Incubation, Control

Journal: bioRxiv

Article Title: Metformin prevented high glucose-induced endothelial reactive oxygen species via OGG1 in an AMPKα-Lin-28 dependent pathway

doi: 10.1101/2020.11.18.388462

Figure Lengend Snippet: The mRNA stability of OGG1 triggered by metformin was dependent on AMPKα. (A-B) The phosphorylation of AMPKα and total AMPKα were measured after metformin-treated HUVECs. (C-E) OGG1 protein and mRNA levels were tested by WB and qPCR after metformin and high glucose treatment simultaneously with silencing of AMPKα or not in the HUVECs. (F-G) HUVECs were digested and incubated with DHE probe (1 μM) for 30 minutes after silencing AMPKα or not in the presence with HG and metformin. The mean intensities were obtained via flow Jo software. Scar bar =25μm. *P<0.05 versus control group. #P<0.05 versus HG plus metformin group. Values are the means±SD with N = 3.

Article Snippet: Primary antibodies anti-OGG1 (NB100-106, Novus Biologicals), anti-Lin28 ser 200 (20607, CST), anti-Lin28 (3695, CST), anti-AMPK (5831, CST), anti-Phospho-AMPKα (50081, CST), and ß-Actin (4970, CST) were obtained commercially.

Techniques: Phospho-proteomics, Incubation, Software, Control

Journal: bioRxiv

Article Title: Metformin prevented high glucose-induced endothelial reactive oxygen species via OGG1 in an AMPKα-Lin-28 dependent pathway

doi: 10.1101/2020.11.18.388462

Figure Lengend Snippet: Lin28 meditated OGG mRNA stability of OGG1 triggered by metformin. (A-B) The subcellular localization of Lin28 were measured after metformin-treated HUVECs in the presence of high glucose. (C-E) OGG1 protein and mRNA levels were tested by WB and qPCR after metformin and high glucose treatment simultaneously with silencing of Lin28 or not in the HUVECs. (F) The half-time hours of OGG1 mRNA was measured after silencing Lin-28 or not with actinomycin D (Act D, 10 μg/ml). *P<0.05 versus control group. Values are the means±SD with N = 3.

Article Snippet: Primary antibodies anti-OGG1 (NB100-106, Novus Biologicals), anti-Lin28 ser 200 (20607, CST), anti-Lin28 (3695, CST), anti-AMPK (5831, CST), anti-Phospho-AMPKα (50081, CST), and ß-Actin (4970, CST) were obtained commercially.

Techniques: Control

Journal: bioRxiv

Article Title: Metformin prevented high glucose-induced endothelial reactive oxygen species via OGG1 in an AMPKα-Lin-28 dependent pathway

doi: 10.1101/2020.11.18.388462

Figure Lengend Snippet: OGG1 expression induced by metformin was dependent on AMPKα phosphorylation of Lin-28. (A-B) The subcellular localization of Lin28 were measured after AMPKα silence or inhibitor pretreatment. *P<0.05 versus HG group. #P<0.05 versus HG plus metformin group. (C-D) OGG1 protein and mRNA levels were tested after silence Lin28 in the HUVECs with AMPKα plasmid or AICAR. *P<0.05 versus HG group. #P<0.05 versus HG plus ACICAR group. Values are the means±SD with N = 3.

Article Snippet: Primary antibodies anti-OGG1 (NB100-106, Novus Biologicals), anti-Lin28 ser 200 (20607, CST), anti-Lin28 (3695, CST), anti-AMPK (5831, CST), anti-Phospho-AMPKα (50081, CST), and ß-Actin (4970, CST) were obtained commercially.

Techniques: Expressing, Phospho-proteomics, Plasmid Preparation

Journal: bioRxiv

Article Title: Metformin prevented high glucose-induced endothelial reactive oxygen species via OGG1 in an AMPKα-Lin-28 dependent pathway

doi: 10.1101/2020.11.18.388462

Figure Lengend Snippet: The protective role of metformin on endothelial ROS was dependent on AMPKα/Lin-28/OGG1 pathway. (A-B) Endothelial ROS levels were assessed by DHE after Compound C or silence of Lin-28-treated HUVECs. *P<0.05 versus HG group. #P<0.05 versus HG plus metformin (Met) group. Values are the means±SD with N = 3.

Article Snippet: Primary antibodies anti-OGG1 (NB100-106, Novus Biologicals), anti-Lin28 ser 200 (20607, CST), anti-Lin28 (3695, CST), anti-AMPK (5831, CST), anti-Phospho-AMPKα (50081, CST), and ß-Actin (4970, CST) were obtained commercially.

Techniques:

Journal: bioRxiv

Article Title: Metformin prevented high glucose-induced endothelial reactive oxygen species via OGG1 in an AMPKα-Lin-28 dependent pathway

doi: 10.1101/2020.11.18.388462

Figure Lengend Snippet: OGG1 regulated HG-induced endothelial ROS via NFκB/NOX4 pathway. (A-B) NADPH oxidases were measured after HUVECs exposure to high glucose treatment with overexpression of OGG1. (B) was statistical result of (A). (C-D) NFκB activity was measured after OGG1 overexpression with high glucose treatment for 24 hours. (E-F) NOX4 protein level was assessed after NFκB silence with high glucose incubation. *P<0.05 versus Ctrl group. #P<0.05 versus HG group. NS P means no difference. Values are the means±SD with N = 3.

Article Snippet: Primary antibodies anti-OGG1 (NB100-106, Novus Biologicals), anti-Lin28 ser 200 (20607, CST), anti-Lin28 (3695, CST), anti-AMPK (5831, CST), anti-Phospho-AMPKα (50081, CST), and ß-Actin (4970, CST) were obtained commercially.

Techniques: Over Expression, Activity Assay, Incubation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Excessive iron inhibits insulin secretion via perturbing transcriptional regulation of SYT 7 by OGG1

doi: 10.1007/s00018-023-04802-y

Figure Lengend Snippet: OGG1/SYT7 is indispensable for INS secretion. A–C A The SYT7 level in Ogg1-knockdown MIN6 cells with SYT7 overexpression. B The gray analysis statistical value of three independent experiments. C Intracellular INS level in Ogg1-knockdown MIN6 cells with SYT7 overexpression. Data are presented as mean ± s.d. of three separate experiments. ***P < 0.001. D The content of INS in cell culture supernatant from Ogg1-knockdown MIN6 cells with SYT7 overexpression. Data are presented as mean ± s.d. of three separate experiments. ***P < 0.001. E–G E Syt7 level of Ogg1-knockdown MIN6 cells treated with GLP-1 were detected by Western blot. F The gray analysis statistical value of three independent experiments. G Intracellular INS level of Ogg1-knockdown MIN6 cells treated with GLP-1 were detected by ELISA. Data are presented as mean ± s.d. of three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001. H The INS level in the supernatant of the cell culture medium of Ogg1-knockdown MIN6 cells treated with GLP-1 was detected by ELISA. Data are presented as mean ± s.d. of three separate experiments. *P < 0.05, **P < 0.01. I–K I The intracellular ultrastructure of MIN6 and Beta-TC-6 cells. Scale bar 1 μm. J The average INS granules diameter in cells of each group. K Number of PM docked granules in each group. Data are presented as mean ± s.d. of three separate experiments. *P < 0.05, **P < 0.01. L Serum basal INS levels in Ogg1−/− mice 3 weeks after AAV8-SYT7 injection. n = 5 in each cohort. **P < 0.01, ***P < 0.001. M, N Hyperglycemic clamp tests show INS biphasic secretion and AUC in Ogg1−/− mice 3 weeks after AAV8-SYT7 injection. 0–10 min is defined as the first phase and 10–30 min is defined as the second phase. n = 3 in each cohort. *Indicates the Ogg1−/−-AAV8-null group compared with the Ogg1+/+-AAV8-null group, *P < 0.05, **P < 0.01, ***P < 0.001. #Indicates the Ogg1−/−-AAV8-SYT7 group compared with the Ogg1−/−-AAV8-null group, #P < 0.05, ##P < 0.01. In the comparison of the AUC of each group, **P < 0.01, ***P < 0.001. O GSIS levels of isolated islets from Ogg1−/− mice 3 weeks after AAV8-SYT7 injection. n = 5 in each cohort. **P < 0.01, ***P < 0.001. P, Q The expression of the AAV8 vector tag GFP and Syt7 in the islets of the Ogg1+/+-AAV8-null, Ogg1−/−-AAV8-null and Ogg1−/−-AAV8-SYT7 mice 3 weeks after AAV8-SYT7 injection were detected by immunofluorescence. Scale bar 20 μm. R INS content in isolated islets from Ogg1−/− mice 3 weeks after AAV8-SYT7 injection. n = 5 in each cohort. *P < 0.05, **P < 0.01. S, T Ogg1−/−-AAV8-SYT7 mice 3 weeks after AAV8-SYT7 injection, the results of the IPGTT experiment and the corresponding AUC. n = 5 in each cohort. For S, *Indicates the Ogg1−/−-AAV8-null group compared with the Ogg1+/+-AAV8-null group, ***P < 0.001. #Indicates the Ogg1−/−-AAV8-SYT7 group compared with the Ogg1−/−-AAV8-null group, ###P < 0.001. In the comparison of the AUC of each group, ***P < 0.001. U, V Ogg1−/−-AAV8-SYT7 mice 3 weeks after AAV8-SYT7 injection, the results of the IPITT experiment and the corresponding AUC. n = 5 in each cohort. ns, no significant difference

Article Snippet: The primary antibodies anti-OGG1 (sc-376835) and anti-synaptotagmin VII (sc-293343) were from Santa Cruz (diluted 1:200) and anti-INS (ab181547) was from Abcam (diluted 1:1000) and Anti-β-tubulin was from Abgent (AM1031A, diluted 1:1000).

Techniques: Knockdown, Over Expression, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Comparison, Isolation, Expressing, Plasmid Preparation, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Excessive iron inhibits insulin secretion via perturbing transcriptional regulation of SYT 7 by OGG1

doi: 10.1007/s00018-023-04802-y

Figure Lengend Snippet: OGG1 deficiency impaired INS secretion. A Weight changes of the Ogg1−/− and Ogg1+/+ mice aged 4–20 weeks. n = 6 in each cohort. *P < 0.05, **P < 0.01. B Random blood glucose of the Ogg1−/− and Ogg1+/+ mice aged 4–20 weeks. n = 6 in each cohort. *P < 0.05, **P < 0.01. C Serum INS content in the Ogg1−/− and Ogg1+/+ mice aged 6 weeks, n = 13 in each cohort. ***P < 0.001. D, E The results of the Ogg1−/− and Ogg1+/+ mice aged 6 weeks detected by IPGTT and the corresponding AUC. n = 5 in each cohort. **P < 0.01, ***P < 0.001. F, G The results of the Ogg1−/− and Ogg1+/+ mice aged 6 weeks detected by IPITT and the corresponding AUC. n = 5 in each cohort. ns, no significant difference. H, I H The levels of Ogg1 in islets of the Ogg1−/− and Ogg1+/+ mice aged 6 weeks were detected by immunofluorescence. I The levels of INS in islets of the Ogg1−/− and Ogg1+/+ mice aged 6 weeks were detected by ELISA. n = 5 in each cohort. *P < 0.05. Scale bar 20 μm. J, K Hyperglycemic clamp tests show INS biphasic secretion and AUC in Ogg1−/− and Ogg1+/+ mice aged 6 weeks. 0–10 min is defined as the first phase and 10–30 min is defined as the second phase. n = 3 in each cohort. *P < 0.05, **P < 0.01, ***P < 0.001. L GSIS and KSIS levels in islets of the Ogg1−/− and Ogg1+/+ mice aged 6 weeks. Data are presented as mean ± s.d. of three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001. M The INS level in the supernatant of MIN6 cells after knockdown of Ogg1 were detected by ELISA. Data are presented as mean ± s.d. of six separate experiments. ***P < 0.001. N–P N Ogg1 knockdown in MIN6 cell line and Ogg1 content. O, the gray analysis statistical value of three independent experiments. P detection of intracellular INS level after shOgg1 by ELISA. Q Cell viability of MIN6 cells with Ogg1 knockdown was detected by CCK8 assay. R The effect of knocking down Ogg1 and using OGG1 inhibitor TH5487 on GSIS and KSIS levels in MIN6 cells. S The INS secretion level of MIN6 with OGG1 inhibitor TH5487 treatment were detected by ELISA. T–V T The Ogg1 content of MIN6 cells with or without Th5487 treatment. U The gray analysis statistical value of three independent experiments. V ELISA results show the fold changes in intracellular INS levels after Th5487 pretreatment compared to DMSO. N–V Data are presented as mean ± s.d. of three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The primary antibodies anti-OGG1 (sc-376835) and anti-synaptotagmin VII (sc-293343) were from Santa Cruz (diluted 1:200) and anti-INS (ab181547) was from Abcam (diluted 1:1000) and Anti-β-tubulin was from Abgent (AM1031A, diluted 1:1000).

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Knockdown, CCK-8 Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Excessive iron inhibits insulin secretion via perturbing transcriptional regulation of SYT 7 by OGG1

doi: 10.1007/s00018-023-04802-y

Figure Lengend Snippet: SYT7 is a new downstream target gene of OGG1. A, B The Ogg1+/+ and Ogg1−/− MEF gene microarray test results. n = 3 in each cohort. C, D Gene expression in MIN6 cells after knockdown of Ogg1. Data are presented as mean ± s.d. of three separate experiments. **P < 0.01, ***P < 0.001. ns no significant difference. E, F Gene expression in MIN6 cells after overexpression of OGG1. Data are presented as mean ± s.d. of three separate experiments. *P < 0.05, ***P < 0.001, ns no significant difference. G RNA-Seq data in the GEO database show the correlation between OGG1 and SYT7 expression in human β cells (data from GEO: GSE165121). Spearman related analysis. P = 0.015. H Double luciferase reporter system was used to detect the effect of Ogg1 knockdown, TH5497 and OGG1 overexpression on the fluorescence signal intensity of pGL3-Syt7. Data are presented as mean ± s.d. of three separate experiments. ***P < 0.001

Article Snippet: The primary antibodies anti-OGG1 (sc-376835) and anti-synaptotagmin VII (sc-293343) were from Santa Cruz (diluted 1:200) and anti-INS (ab181547) was from Abcam (diluted 1:1000) and Anti-β-tubulin was from Abgent (AM1031A, diluted 1:1000).

Techniques: Microarray, Gene Expression, Knockdown, Over Expression, RNA Sequencing, Expressing, Luciferase, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Excessive iron inhibits insulin secretion via perturbing transcriptional regulation of SYT 7 by OGG1

doi: 10.1007/s00018-023-04802-y

Figure Lengend Snippet: Excessive iron inhibited OGG1 binding to the SYT7 promoter. A, B Immunofluorescence staining of Ogg1, INS and Syt7 proteins in mouse β cells with FeSO4 intake for 2–4 months. Scale bar 20 μm. C The relative expression of the Syt7 gene in the MIN6 cells treated with FeSO4 for 1, 2, 3, 4, 5, 9, and 11 days. Data are presented as mean ± s.d. of three separate experiments. *P < 0.05, ***P < 0.001. D, E The expression of Ogg1 and Syt7 in mouse β cells were detected by RNA-seq. (data from GEO: GSE128945). n = 3 in each cohort. **P < 0.01, ***P < 0.001. E Person related analysis. P = 0.001. F EMSA shows the binding of OGG1 to the SYT7 promoter after FeSO4 (100 μM) treatment. G ChIP-qPCR analysis of Ogg1 binding to the Syt7 promoter in MIN6 cells treated with FeSO4. Data are presented as mean ± s.d. of three separate experiments. ***P < 0.001. H Dual luciferase reporter analysis of the effect of FeSO4 treatment on Syt7 promoter activity in 293 T cells with OGG1 overexpression. Firefly luciferase activity was measured 24 h post transfection and normalized to Renilla luciferase activity. Data are presented as mean ± s.d. of three separate experiments. ***P < 0.001. I, J Fe2+-4m mice 3 weeks after AAV8-SYT7 or Trulicity injection, the results of the IPGTT experiment and the corresponding AUC. n = 5 in each cohort. *Indicates the Fe2+-AAV8-null group compared with the CW-AAV8-null group, *P < 0.05, ***P < 0.001. #Indicates that the Fe2+-AAV8-SYT7 group was compared with the Fe2+-AAV8-null group. #P < 0.05, ##P < 0.01, ###P < 0.001. aIndicates that the Fe2+-Trulicity group was compared with the Fe2+-AAV8-SYT7 group, aP < 0.05, aaP < 0.01, aaaP < 0.001. In the comparison of the AUC of each group, ***P < 0.001. K, L Fe2+-4m mice 3 weeks after AAV8-SYT7 or Trulicity injection, the results of IPITT experiment and corresponding AUC. n = 5 in each cohort. *Indicates the Fe2+-AAV8-null group compared with the CW-AAV8-null group, *P < 0.05, **P < 0.01, ***P < 0.001. #Indicates that the Fe2+-AAV8-SYT7 group was compared with the Fe2+-AAV8-null group. ##P < 0.01. aIndicates that the Fe2+-Trulicity group was compared with the Fe2+-AAV8-SYT7 group, aP < 0.05. In the comparison of the AUC of each group, ***P < 0.001, ns no significant difference. M Serum basal INS content of Fe2+-4m mice 3 weeks after AAV8-SYT7 or Trulicity injection. n = 5 in each cohort. **P < 0.01, ***P < 0.001. ns, no significant difference. N, O Hyperglycemic clamp tests show INS biphasic secretion and AUC of Fe2+-4m mice 3 weeks after AAV8-SYT7 or Trulicity injection. 0–10 min is defined as the first phase and 10–30 min is defined as the second phase. n = 3 in each cohort. N *Indicates the Fe2+-AAV8-null group compared with the CW-AAV8-null group, *P < 0.05, **P < 0.01, ***P < 0.001. #Indicates the Fe2+-AAV8-SYT7 group compared with the Fe2+-AAV8-null group, #P < 0.05, ##P < 0.01. aIndicates that the Fe2+-Trulicity group was compared with the Fe2+-AAV8-SYT7 group, aaP < 0.01. In the comparison of the AUC of each group, **P < 0.01, ***P < 0.001. P Effect of AAV8-SYT7 injection on GSIS of isolated islets in each group. n = 5 in each cohort. *P < 0.05, ***P < 0.001. Q After 3 weeks of Fe2+-4m mice 3 weeks injected with AAV8-SYT7 or Trulicity, immunofluorescence show the staining of Syt7 in the β cells of each group. Scale bar 20 μm. R The effect of AAV8-SYT7 on the INS content in islets in each group was detected by ELISA. n = 5 in each cohort. **P < 0.01, ***P < 0.001. S–U S The intracellular ultrastructure of MIN6 and Beta-TC-6 Fe2+ model after SYT7 overexpression. Scale bar 1 μm. T The average INS granules diameter in cells of each group. U Number of PM docked granules in each group. Data are presented as mean ± s.d. of three separate experiments. *P < 0.05, **P < 0.01

Article Snippet: The primary antibodies anti-OGG1 (sc-376835) and anti-synaptotagmin VII (sc-293343) were from Santa Cruz (diluted 1:200) and anti-INS (ab181547) was from Abcam (diluted 1:1000) and Anti-β-tubulin was from Abgent (AM1031A, diluted 1:1000).

Techniques: Binding Assay, Immunofluorescence, Staining, Expressing, RNA Sequencing, ChIP-qPCR, Luciferase, Activity Assay, Over Expression, Transfection, Injection, Comparison, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Excessive iron inhibits insulin secretion via perturbing transcriptional regulation of SYT 7 by OGG1

doi: 10.1007/s00018-023-04802-y

Figure Lengend Snippet: Schematic diagram demonstrating INS secretion upon glucose stimulation. Left panel: β cell responds to glucose signal when the Fe2+ level is maintained in a constant range. OGG1 binds to SYT7 promoter and upregulates SYT7 expression, the latter is required for transport vesicles formation and INS secretion. Right panel: excessive iron disrupts the transcriptional regulation of SYT7 by OGG1, reduces the number of INS transport vesicles, agglomerates INS granules and inhibits INS secretion

Article Snippet: The primary antibodies anti-OGG1 (sc-376835) and anti-synaptotagmin VII (sc-293343) were from Santa Cruz (diluted 1:200) and anti-INS (ab181547) was from Abcam (diluted 1:1000) and Anti-β-tubulin was from Abgent (AM1031A, diluted 1:1000).

Techniques: Expressing