Journal: International Journal of Molecular Medicine

Article Title: Metformin reverses oxidative stress-induced mitochondrial dysfunction in pre-osteoblasts via the EGFR/GSK-3β/calcium pathway

doi: 10.3892/ijmm.2023.5239

Figure Lengend Snippet: RNA interference sequences used for transfection.

Article Snippet: Primary antibodies against GSK-3β (cat. no. 12456), p-GSK-3β (cat. no. 5558), cleaved caspase-3 (cat. no. 9661), and β-actin (cat. no. 4970) were obtained from Cell Signaling Technology, Inc.

Techniques: Transfection

Journal: International Journal of Molecular Medicine

Article Title: Metformin reverses oxidative stress-induced mitochondrial dysfunction in pre-osteoblasts via the EGFR/GSK-3β/calcium pathway

doi: 10.3892/ijmm.2023.5239

Figure Lengend Snippet: Metformin improves pre-osteoblast apoptosis induced by oxidative stress through GSK-3β. (A) The localization and expression of GSK-3β was observed in MC3T3-E1 cells and GSK-3β was in a more aggregated state. (B) The silencing efficiency of GSK-3β was detected at the protein level. (C) The apoptotic rate after the silencing of GSK-3β was detected. The apoptotic rate of the H 2 O 2 group was 32.77%, that of the H 2 O 2 + metformin group was 10.81%, and that of the H 2 O 2 + metformin + siRNA group was 23.52%. (D) The level of ROS in the cytoplasm was detected before and after treatment with metformin and siRNA-GSK-3β. (E) The fluorescence intensity of ROS was detected using flow cytometry. (F) The expression of mitochondrial apoptosis-related proteins after the silencing of GSK-3β was detected again. The experiments were performed three times. Data are presented as the mean ± SD. ** P<0.01, compared with the control cells; ## P<0.01, compared with the H 2 O 2 group; ^^ P<0.01, compared with the H 2 O 2 + Met group. ns, not significant; ROS, reactive oxygen species; Met, metformin; H 2 O 2 , hydrogen peroxide; GSK-3β, glycogen synthase kinase 3β.

Article Snippet: Primary antibodies against GSK-3β (cat. no. 12456), p-GSK-3β (cat. no. 5558), cleaved caspase-3 (cat. no. 9661), and β-actin (cat. no. 4970) were obtained from Cell Signaling Technology, Inc.

Techniques: Expressing, Fluorescence, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Metformin reverses oxidative stress-induced mitochondrial dysfunction in pre-osteoblasts via the EGFR/GSK-3β/calcium pathway

doi: 10.3892/ijmm.2023.5239

Figure Lengend Snippet: Metformin inhibits mPTP opening and Ca2+-mediated mitochondrial dysfunction via GSK-3β. (A) The effect of Met on mitochondrial membrane potential in MC3T3-E1 cells was detected. (B) The ratio of red/green fluorescence representing mitochondrial membrane potential was quantitatively analyzed using flow cytometry. (C) Intracellular calcium was observed using a fluorescence electron microscope. (D) Mitochondrial superoxide was observed using a fluorescence electron microscope. Blue fluorescence indicates DAPI, and red fluorescence indicates mitochondrial superoxide. (E) Quantitative analysis of calcium fluorescence intensity using flow cytometry. (F) Quantitative analysis of fluorescence intensity of mitochondrial superoxide using flow cytometry. (G) Quantitative analysis of the levels of calcium. (H) Quantitative analysis of the levels of mitochondrial superoxide. The experiments were performed three times. Data are presented as the mean ± SD. * P<0.05 and ** P<0.01, compared with the control cells; ## P<0.01, compared with the H 2 O 2 group; ^^ P<0.01, compared with the H 2 O 2 + Met group. ns, not significant; Met, metformin; H 2 O 2 , hydrogen peroxide; GSK-3β, glycogen synthase kinase 3β.

Article Snippet: Primary antibodies against GSK-3β (cat. no. 12456), p-GSK-3β (cat. no. 5558), cleaved caspase-3 (cat. no. 9661), and β-actin (cat. no. 4970) were obtained from Cell Signaling Technology, Inc.

Techniques: Fluorescence, Flow Cytometry, Microscopy

Journal: International Journal of Molecular Medicine

Article Title: Metformin reverses oxidative stress-induced mitochondrial dysfunction in pre-osteoblasts via the EGFR/GSK-3β/calcium pathway

doi: 10.3892/ijmm.2023.5239

Figure Lengend Snippet: Metformin reverses pre-osteoblast apoptosis induced by oxidative stress by phosphorylating GSK-3β via the EGFR pathway. (A) The effect of metformin on the phosphorylation level of GSK-3β was detected at the protein level. (B) The KEGG pathway of differentially expressed genes in OVX vs. OVX + Met. The EGFR pathway has a lower P-value and better enrichment. OVX represents post-menopausal osteoporosis mice. (C) The expression and regulation of EGFR was examined using gene set enrichment analysis. (D) The phosphorylation level of EGFR was detected at the protein level. GSK-3β, glycogen synthase kinase 3β.

Article Snippet: Primary antibodies against GSK-3β (cat. no. 12456), p-GSK-3β (cat. no. 5558), cleaved caspase-3 (cat. no. 9661), and β-actin (cat. no. 4970) were obtained from Cell Signaling Technology, Inc.

Techniques: Expressing

Journal: International Journal of Molecular Medicine

Article Title: Metformin reverses oxidative stress-induced mitochondrial dysfunction in pre-osteoblasts via the EGFR/GSK-3β/calcium pathway

doi: 10.3892/ijmm.2023.5239

Figure Lengend Snippet: Flow chart of metformin regulating the mPTP of MC3T3-E1 cells through the EGFR/GSK-3β/calcium axis and reversing osteoporosis caused by oxidative stress. ROS, reactive oxygen species; GSK-3β, glycogen synthase kinase 3β; mPTP, mitochondrial permeability transition pore.

Article Snippet: Primary antibodies against GSK-3β (cat. no. 12456), p-GSK-3β (cat. no. 5558), cleaved caspase-3 (cat. no. 9661), and β-actin (cat. no. 4970) were obtained from Cell Signaling Technology, Inc.

Techniques: Permeability

Journal: International Journal of Oncology

Article Title: A disintegrin and metalloprotease 12 contributes to colorectal cancer metastasis by regulating epithelial-mesenchymal transition

doi: 10.3892/ijo.2023.5498

Figure Lengend Snippet: Effects of ADAM12 on oncogenic signaling pathways in the human colorectal cancer cells. Representative western blot images and semi-quantification graphs for the phosphorylation levels of PDK1, GSK-3β and AKT in AV-transfected or AS-transfected DLD1 or SW480 cells. Data are presented as the mean ± SD (n=3). * P<0.05. ADAM12, a disintegrin and metalloprotease 12; PDK1, phosphoinositide-dependent protein kinase 1; GSK-3β, glycogen synthase kinase-3β; EV, empty pcDNA6-myc vector; AV, ADAM12-pcDNA6-myc construct; SS, scrambled siRNA; AS, ADAM12 siRNA; siRNA, small interfering RNA; p-, phosphorylated; T-, total.

Article Snippet: Antibodies against E-cadherin (cat. no. #14472), Snail (cat. no. #3879), vimentin (cat. no. #5741), claudin-1 (cat. no. #4933), integrin α5 (cat. no. #4705), integrin β1 (cat. no. #9699), integrin β3 (cat. no. #13166), phosphorylated (p)-AKT (S473) (cat. no. #4060), p-phosphoinositide-dependent protein kinase 1 (PDK1) (S241) (cat. no. #3438), p-glycogen synthase kinase-3β (GSK-3β) (S9) (cat. no. #9323), total AKT (cat. no. #4691), total PDK1 (cat. no. #3062), total GSK-3β (cat. no. #9832) and Myc-tag (cat. no. #2278) were obtained from Cell Signaling Technology, Inc. Antibodies against β-tubulin (cat. no. sc-9104) and GAPDH (cat. no. sc-25778) were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Western Blot, Transfection, Plasmid Preparation, Construct, Small Interfering RNA

Journal: medRxiv

Article Title: Reducing squalene epoxidase by the aging-dependent intra-tissue cholesterol accumulation is associated with increased colorectal cancer patient severity in high-risk populations

doi: 10.1101/2023.03.28.23287791

Figure Lengend Snippet: Involvement of SQLE, p53 WT , p53 MT , and GSK3β pS9 in aging-dependent CRC progression. Each CRC specimen grouped according to patient age (A-D) or CRC grade (E) was stained simultaneously with antibodies against the indicated candidate and then observed under confocal microscopy. The quantification of each variable shown in (A) and (C) was presented in (B) and (D), respectively . The tissues were supplied by TissueArray (A-B, E) or Chungnam National University Hospital (C-D). p53 WT , mainly detecting wild-type p53 (DO-1), p53 MT ; mainly mutant p53 (Y5), and GSK3β pS9 : the inactive form of GSK3β determined by the anti-GSK3β pS9 antibody. Scale bar, 100 μm. * An unpaired t-test determined P values.

Article Snippet: Briefly, deparaffinized and antigen-retrieved slides were reacted with antibodies against SQLE, p53 WT , GSK3β (Santa Cruz Biotechnology, Dallas, TX, USA; SC-99144, SC-126, SC-377213, respectively), p53 MT (Abcam, Boston, MA, USA; ab32049), and GSK3β pS9 (Cell Signaling Technology, Danvers, MA, USA; 9323s) overnight.

Techniques: Staining, Confocal Microscopy, Mutagenesis

Journal: medRxiv

Article Title: Reducing squalene epoxidase by the aging-dependent intra-tissue cholesterol accumulation is associated with increased colorectal cancer patient severity in high-risk populations

doi: 10.1101/2023.03.28.23287791

Figure Lengend Snippet: Relevance of cholesterol augmentation in age-related CRC progression. The total cholesterol (A) and cholesteryl ester (B) in CRC tissue lysates were measured according to the manufacturer’s instructions. Dots indicate a single CRC sample. (C) For Western blot analysis, ten independent human CRC specimens were used. For loading control, GAPDH was used. (D) ImageJ (National Institute of Health, Bethesda, Maryland, USA) was used to quantify protein expression, r.u, relative uncertainty, and P values were calculated by an unpaired t-test. p53 WT , mainly detecting wild-type p53 (DO-1); p53 MT , mainly to detect mutant p53 (Y5); GSK3β pS9 , the inactive form of GSK3β.

Article Snippet: Briefly, deparaffinized and antigen-retrieved slides were reacted with antibodies against SQLE, p53 WT , GSK3β (Santa Cruz Biotechnology, Dallas, TX, USA; SC-99144, SC-126, SC-377213, respectively), p53 MT (Abcam, Boston, MA, USA; ab32049), and GSK3β pS9 (Cell Signaling Technology, Danvers, MA, USA; 9323s) overnight.

Techniques: Western Blot, Expressing, Mutagenesis

Journal: medRxiv

Article Title: Reducing squalene epoxidase by the aging-dependent intra-tissue cholesterol accumulation is associated with increased colorectal cancer patient severity in high-risk populations

doi: 10.1101/2023.03.28.23287791

Figure Lengend Snippet: Multivariate analysis of the candidates and pathological factors related to survival in an independent set generated from the discovery cohort by applying random forest and then separated by before and after age 50. (a) p53 WT : wild-type p53 (DO-1), (b) p53 MT : mutant p53 (Y5), (c) GSK3β pS9 : the inactive form of GSK3β (the anti-GSK3β pS9 antibody), (d) Sex (Male): risk assessment for males compared to females, and (e) T3N0M0 including T1N0M0 and T2N0M0. P values are noted on the dashed line of HR weights. (A) Training and (B) testing sets.

Article Snippet: Briefly, deparaffinized and antigen-retrieved slides were reacted with antibodies against SQLE, p53 WT , GSK3β (Santa Cruz Biotechnology, Dallas, TX, USA; SC-99144, SC-126, SC-377213, respectively), p53 MT (Abcam, Boston, MA, USA; ab32049), and GSK3β pS9 (Cell Signaling Technology, Danvers, MA, USA; 9323s) overnight.

Techniques: Generated, Mutagenesis

Journal: medRxiv

Article Title: Reducing squalene epoxidase by the aging-dependent intra-tissue cholesterol accumulation is associated with increased colorectal cancer patient severity in high-risk populations

doi: 10.1101/2023.03.28.23287791

Figure Lengend Snippet: Time-dependent ROC analysis for prognostic patient survival within a given time. The time was decided to maximize the ROC curve utilizing a weighted Cox regression. (A-B) Training and (C-D) testing sets, and the times for patients before (A, C) and after (B, D) age 50 were 123 and 137, respectively. p53 WT , mainly detecting wild-type p53 (DO-1), p53 MT ; to detect mutant p53 (Y5), and GSK3β pS9 : the inactive form of GSK3β (the anti-GSK3β pS9 antibody).

Article Snippet: Briefly, deparaffinized and antigen-retrieved slides were reacted with antibodies against SQLE, p53 WT , GSK3β (Santa Cruz Biotechnology, Dallas, TX, USA; SC-99144, SC-126, SC-377213, respectively), p53 MT (Abcam, Boston, MA, USA; ab32049), and GSK3β pS9 (Cell Signaling Technology, Danvers, MA, USA; 9323s) overnight.

Techniques: Mutagenesis

Journal: medRxiv

Article Title: Reducing squalene epoxidase by the aging-dependent intra-tissue cholesterol accumulation is associated with increased colorectal cancer patient severity in high-risk populations

doi: 10.1101/2023.03.28.23287791

Figure Lengend Snippet: Diagnostic analysis using the discriminant scores for each candidate as a single or whole. The receiver operating characteristic (ROC) curves of the discriminant scores for each candidate in the (A) training and (B) testing sets are shown. (C-F) Each set is divided into before (C, E) and after (D, F) age 50; a linear discriminant distribution for merged candidates (left) and the ROC analysis using the discriminant score for the merged candidates (right) for diagnosing CRC are represented. The ratio of AUC and the corresponding 95% CI are noted. p53 WT ; wild-type p53 (DO-1), p53 MT ; mutant p53 (Y5), and GSK3β pS9 : the inactive form of GSK3β determined by the anti-GSK3β pS9 antibody. (A, C, D) Training and (B, E, F) testing sets.

Article Snippet: Briefly, deparaffinized and antigen-retrieved slides were reacted with antibodies against SQLE, p53 WT , GSK3β (Santa Cruz Biotechnology, Dallas, TX, USA; SC-99144, SC-126, SC-377213, respectively), p53 MT (Abcam, Boston, MA, USA; ab32049), and GSK3β pS9 (Cell Signaling Technology, Danvers, MA, USA; 9323s) overnight.

Techniques: Diagnostic Assay, Mutagenesis

Journal: eLife

Article Title: Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities

doi: 10.7554/eLife.78629

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-pGSK3β-Ser9 (rabbit polyclonal) , Cell Signaling Technology , Cat# 5558; RRID :AB_10013750 , WB (1:500).

Techniques: Knock-Out, Transgenic Assay, Recombinant, Plasmid Preparation, Clone Assay, Sequencing, DC Protein Assay, Transformation Assay, Isolation, Protease Inhibitor, Western Blot, Software

Journal: Antioxidants

Article Title: Inhibiting NLRP3 Inflammasome Activation by CY-09 Helps to Restore Cerebral Glucose Metabolism in 3×Tg-AD Mice

doi: 10.3390/antiox12030722

Figure Lengend Snippet: CY-09 reversed the expression and distribution of tau-related proteins in 3×Tg-AD mice. ( a – e ) Western blot analysis of Tau5, HT7, p-Tau-Ser404, GSK3β, and p-GSK3β-Ser9 in NTg, NTg+CY-09, 3×Tg-AD, and 3×Tg-AD + CY-09 mice. (n = 6, mean ± SD, one-way ANOVA and Bonferroni post hoc test; *** p < 0.001 vs. NTg mice, & p < 0.05, && p < 0.01, &&& p < 0.001 vs. NTg + CY-09 mice, # p < 0.05 vs. 3×Tg-AD mice). ( f ) Distribution of p-Tau-Ser404 in the CA3 region and cortex of the four groups of mice (scale bar: 100 μm).

Article Snippet: Antibodies against IRS (Cat#3407), p-IRS-Ser1101 (Cat#2385), AKT (Cat#9272), p-AKT-Ser473 (Cat#4060), GSK3β (Cat#12456), p-GSK3β-Ser9 (Cat#5558), FAS (Cat#3180), ACC (Cat#3676), p-ACC-Ser79 (Cat#11818), and LRP1 (Cat#64099) were purchased from Cell Signaling Technology, Inc., Boston, MA, USA.

Techniques: Expressing, Western Blot

Journal: Antioxidants

Article Title: Different Effects and Mechanisms of Selenium Compounds in Improving Pathology in Alzheimer’s Disease

doi: 10.3390/antiox12030702

Figure Lengend Snippet: Antibodies.

Article Snippet: Rabbit anti-pGSK3β (Ser9) , CST , 9323.

Techniques:

Journal: Antioxidants

Article Title: Different Effects and Mechanisms of Selenium Compounds in Improving Pathology in Alzheimer’s Disease

doi: 10.3390/antiox12030702

Figure Lengend Snippet: Levels of key proteins, kinases, and receptors in the signaling pathways related to AD pathologies. ( a ) Levels of GSK-3β and phosphorylated GSK-3β (Ser9) in the cortexes of 3× Tg-AD mouse brains were analyzed by Western blotting. ( b ) Quantification of protein levels in ( a ). ( c ) Levels of COX IV, Drp1, and OPA1 were analyzed by Western blotting. ( d – f ) Quantification of protein levels in ( c ). ( g ) Levels of FoxO6, FoxO3a, and phosphorylated FoxO3a (Ser253) were analyzed by Western blotting. ( h , i ) Quantification of protein levels in ( g ). ( j ) Levels of NMDAR1, NMDAR2A, and NMDAR2B were analyzed by Western blotting. ( k – m ) Quantification of protein levels in ( j ). α-Tubulin, β-actin, or GAPDH were used as loading controls. All data are presented as the means ± SEMs ( n = 3). * p < 0.05 vs. AD mice, as determined by one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: Rabbit anti-pGSK3β (Ser9) , CST , 9323.

Techniques: Western Blot

Journal: International Journal of Stem Cells

Article Title: Mesenchymal Stem Cells Ameliorate Fibrosis by Enhancing Autophagy via Inhibiting Galectin-3/Akt/mTOR Pathway and by Alleviating the EMT via Inhibiting Galectin-3/Akt/GSK3β/Snail Pathway in NRK-52E Fibrosis

doi: 10.15283/ijsc22014

Figure Lengend Snippet: Effects of SF-MSCs-CM on TGF-β 1-induced Gal-3 KD NRK-52E cells and Gal-3 OE NRK-52E cells fibrosis. The black and white bands illustrating the indicators identified through Western Blot (A), and statistical analysis of the ratio relative to GAPDH, including α-MSA (B), the ratio of TIMP1/MMP9 (C), FN (D), E-Cadherin (E), KIM-1 (F), Galectin-3 (G), the ratio of P-Akt/Akt (H) and P-GSK3β/GSK3β1 (I), and Snail (J). SF-MSCs-CM treatment significantly downregulated these indexes more surely than SF-DMEM treatment after TGF-β 1 treatment, except the expression of E-cad, which was upregulated. In the Gal-3 OE group, the indexes presented similar trends to those of the Gal-3 KD group, but higher than those of the same subgroups in the Gal-3 KD group. Results were normalized relative to the expression of GAPDH. N=3 (per group). Data are presented as mean±SD and analyzed by two-way ANOVA followed by Tukey post hoc testing. *p<0.05, vs. control group, ★ p<0.05, vs. TGF-β 1＋SF-MSCs-CM group, ▲ p<0.05, vs. TGF-β 1＋SF-DMEM group, ● p<0.05, vs. SF-MSCs-CM group; compared Gal-3 KD NRK-52E cells with Gal-3 OE NRK-52E cells, # p<0.05, vs. normal group, @ p<0.05, vs. TGF-β 1 group, &p<0.05, vs. TGF-β 1＋SF-MSCs-CM group, ◆ p<0.05, vs. TGF-β 1＋SF-DMEM group, ^p<0.05, vs. SF-MSCs-CM group.

Article Snippet: The following antibodies are used: GAPDH (1：30,000, Proteintech, USA), α-SMA (1：4,000, Proteintech, USA), Galectin-3 (1：3,000, Cell Signaling, USA), GSK3β (1：1,000, Cell Signaling, USA), Phospho-GSK3β (Ser9) (1：1,000, Cell Signaling, USA), Snail (C15D3) (1：1,000, Cell Signaling, USA), Akt (pan) (1：1,000, Cell Signaling, USA), Phospho-Akt (Ser473) (1：1,000, Cell Signaling, USA), Fibronectin (1：1,000, Abcam, UK), E-Cadherin (1：1,000, Abcam, UK), MMP9 (1：1,000, Abcam, UK), TIMP1 (1：1,000, Cell Signaling, USA), Kim-1 (1：1,000, SAB, USA), PI3K (1：1,000, Abmart, China), Phospho-PI3K (1：1,000, Abmart, China), mTOR (1：1,000, Abmart, China), Phospho-mTOR (1：1,000, Abmart, China), LC3B (1：1,000, Abmart, China), and P62 (1：1,000, Abmart, China) antibodies.

Techniques: Western Blot, Expressing