Journal: Antioxidants

Article Title: Tetraselmis chuii Supplementation Increases Skeletal Muscle Nuclear Factor Erythroid 2-Related Factor 2 and Antioxidant Enzyme Gene Expression, and Peak Oxygen Uptake in Healthy Adults: A Randomised Crossover Trial

doi: 10.3390/antiox14040435

Figure Lengend Snippet: Gene names, gene symbols and ThermoFisher Scientific Assay IDs for human skeletal muscle OpenArray™ gene expression analyses.

Article Snippet: p38 Gamma , p38g (MAPK12) , Hs00268060_m1.

Techniques: Gene Expression, Binding Assay

Journal: Antioxidants

Article Title: Tetraselmis chuii Supplementation Increases Skeletal Muscle Nuclear Factor Erythroid 2-Related Factor 2 and Antioxidant Enzyme Gene Expression, and Peak Oxygen Uptake in Healthy Adults: A Randomised Crossover Trial

doi: 10.3390/antiox14040435

Figure Lengend Snippet: Correlations between gene expression in RNA isolated from resting human skeletal muscle following dietary supplementation for 2 weeks with a placebo containing hemicellulose crystalline only compared to the difference in gene expression between placebo and dietary supplementation for 2 weeks with 25 mg/day T. chuii with hemicellulose crystalline as an excipient.

Article Snippet: p38 Gamma , p38g (MAPK12) , Hs00268060_m1.

Techniques: Gene Expression, Isolation, Binding Assay

Journal: Advanced Science

Article Title: Inhibition of Endoplasmic Reticulum Stress Cooperates with SLC7A11 to Promote Disulfidptosis and Suppress Tumor Growth upon Glucose Limitation

doi: 10.1002/advs.202408789

Figure Lengend Snippet: SLC7A11 promotes p38 phosphorylation during disulfidptosis induced by glucose deprivation. A–E) Cell morphological changes (A,C) and cell viability (B,D,E) measured by CCK8 assay in SLC7A11 high UMRC6, H460, and 7402 cells cultured in glucose‐containing (Ctrl) or glucose‐free (‐Glc) medium with or without Z‐VAD (5 µ m ), Nec‐1s (2 µ m ), Liprox‐1 (5 µ m ), CQ (20 µ m ) and NAC (2 m m ) for 4–6 h. Scale bars, 100 µm. F) Western blotting analysis of apoptotic markers in UMRC6 and H460 cells cultured in medium without glucose (Glc) or glutamine (Gln) or with energy stress inducers 2‐DG (10 m m ), metformin (2 m m ) and phenformin (2 m m ) for 8 h. G) Reducing and non‐reducing Western blotting analysis of Drebrin in UMRC6 and 7402 cells cultured in glucose‐containing/‐free medium for 4–6 h. H) Western blotting analysis of SLC7A11 expression in control and SLC7A11 knockout UMRC6 cell lines. I) Antibody microarray analysis of involved signaling molecules in sgCon and sgSLC7A11 UMRC6 cells cultured in glucose‐containing/‐free medium. J) Western blotting analysis of p38 phosphorylation of indicated cells cultured in glucose‐containing/‐free medium. K) Western blotting analysis of SLC7A11 expression and p38 phosphoryation in 786‐O empty vector (EV), wild type (WT), and C86S mutant cell lines cultured in glucose‐containing/‐free medium. L,M) Western blotting analysis of p38 phosphoryation in UMRC6 (L) and 7402 (M) cells cultured in glucose‐containing/‐free medium with or without Cystine, Erastin (10 µ m ) and Sulfasalazine (SAS, 10 µ m ) for 4–6 h. N,O) Cell viability measured by CCK8 assay in UMRC6 (N) and 7402 (O) cells cultured in glucose‐containing/‐free medium with or without indicated concentrations of Cystine, Erastin and SAS for 4–6 h. P) Western blotting analysis of p38 phosphoryation in UMRC6 cells cultured in glucose‐containing/‐free medium with or without p38 inhibitor SB203580 (50 µ m ) for 6 h. Q) Cell viability measured by CCK8 assay in UMRC6 cells cultured in glucose‐containing/‐free medium with or without SB203580 treatment. R) Reducing and non‐reducing Western blotting analysis of Drebrin in UMRC6 and 7402 cells cultured in glucose‐containing/‐free medium with or without SB203580 treatment. S–X) Western blotting analysis of MAPK12 (S) and MAPK14 (W) expression, and RT‐PCR analysis of MAPK13 (U) levels in control and corresponding knockout UMRC6 cell lines. Cell viability was measured by CCK8 assay in sgCon and sgMAPK12/13/14 (T/V/X) UMRC6 cells cultured in glucose‐containing/‐free medium. All p values were calculated using a two‐tailed unpaired Student's t ‐test. Data are mean ± SD, n ≥ 3 independent repeats unless specified. ns: not significant ( p > 0.05). All Western blotting was repeated at least twice, independently, with similar results.

Article Snippet: MAPK12 (#HA722017) and MAPK14 (#ET1602‐26) were obtained from HUABIO (China).

Techniques: Phospho-proteomics, CCK-8 Assay, Cell Culture, Western Blot, Expressing, Control, Knock-Out, Microarray, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Inflammation and Regeneration

Article Title: SPARC activates p38γ signaling to promote PFKFB3 protein stabilization and contributes to keloid fibroblast glycolysis

doi: 10.1186/s41232-024-00357-y

Figure Lengend Snippet: SPARC activated p38γ signaling to stabilize PFKFB3 protein expression. A , B GSEA manifested that SPARC regulated p38 pathway. C The interaction between SPARC, p38γ, and PFKFB3 in PPI network. D After transfected with pcDNA-SPARC, si-SPARC-1#, and si-SPARC-2#, p38γ and PFKFB3 expression was measured via western blot. E After transfected with si-p38γ or treated with SB203580, PFKFB3 expression was measured via western blot. F Western blot of co-IP was performed to verify the binding ability between p38γ and PFKFB3 in KFs. G After transfected with si-p38γ and treated with CHX, p38γ and PFKFB3 expression in KFs was measured via western blot. H Immunohistochemical staining and I western blotting for p38γ and PFKFB3 in human keloids and extra-lesional samples. ** P < 0.01

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the primary antibodies in 5% milk-TBST overnight at 4 °C using the following concentrations: SPARC (1:500, no. ab290636 and ab225716, Abcam, UK), p38γ (1:1000, no. 20184–1-AP, Proteintech, USA), PFKFB3 (1:1000, no. 13763–1-AP, Proteintech, USA), α-SMA (1:1000, no. #19245, Cell Signaling Technology, USA), Fibronectin (1:500, no. ab2413, Abcam, UK), Collagen I (1:500, no. ab138492, Abcam, UK), Collagen III (1:500, no. ab184993, Abcam, UK) and β-actin (1:2000, no. ab8227, Abcam, UK).

Techniques: Expressing, Transfection, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Immunohistochemical staining, Staining

Journal: Inflammation and Regeneration

Article Title: SPARC activates p38γ signaling to promote PFKFB3 protein stabilization and contributes to keloid fibroblast glycolysis

doi: 10.1186/s41232-024-00357-y

Figure Lengend Snippet: SPARC promotes the proliferation, migration, collagen production, and glycolysis of KFs via regulating p38γ signaling. After transfected with pcDNA-SPARC and si-p38γ, and treated with SB203580, ECAR ( A ), OCR ( B ), glucose uptake ( C ), and lactate production ( D ) were investigated in KFs. PFKFB3 expression was measured via western blot ( E ). The proliferation and migration were assessed using EdU ( F ) and transwell assay ( G ). α-SMA, Fibronectin, Collagen I, and Collagen III expression was measured via western blot ( H ). * P < 0.05, ** P < 0.01

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the primary antibodies in 5% milk-TBST overnight at 4 °C using the following concentrations: SPARC (1:500, no. ab290636 and ab225716, Abcam, UK), p38γ (1:1000, no. 20184–1-AP, Proteintech, USA), PFKFB3 (1:1000, no. 13763–1-AP, Proteintech, USA), α-SMA (1:1000, no. #19245, Cell Signaling Technology, USA), Fibronectin (1:500, no. ab2413, Abcam, UK), Collagen I (1:500, no. ab138492, Abcam, UK), Collagen III (1:500, no. ab184993, Abcam, UK) and β-actin (1:2000, no. ab8227, Abcam, UK).

Techniques: Migration, Transfection, Expressing, Western Blot, Transwell Assay

Journal: Inflammation and Regeneration

Article Title: SPARC activates p38γ signaling to promote PFKFB3 protein stabilization and contributes to keloid fibroblast glycolysis

doi: 10.1186/s41232-024-00357-y

Figure Lengend Snippet: SPARC, p38γ, and PFKFB3 were increased in the skin of BLM-induced fibrosis mouse model. A Immunohistochemical staining for SPARC, p38γ, and PFKFB3. B Western blot for assessing SPARC, p38γ, and PFKFB3 expression. ** P < 0.01

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the primary antibodies in 5% milk-TBST overnight at 4 °C using the following concentrations: SPARC (1:500, no. ab290636 and ab225716, Abcam, UK), p38γ (1:1000, no. 20184–1-AP, Proteintech, USA), PFKFB3 (1:1000, no. 13763–1-AP, Proteintech, USA), α-SMA (1:1000, no. #19245, Cell Signaling Technology, USA), Fibronectin (1:500, no. ab2413, Abcam, UK), Collagen I (1:500, no. ab138492, Abcam, UK), Collagen III (1:500, no. ab184993, Abcam, UK) and β-actin (1:2000, no. ab8227, Abcam, UK).

Techniques: Immunohistochemical staining, Staining, Western Blot, Expressing

Journal: Inflammation and Regeneration

Article Title: SPARC activates p38γ signaling to promote PFKFB3 protein stabilization and contributes to keloid fibroblast glycolysis

doi: 10.1186/s41232-024-00357-y

Figure Lengend Snippet: Inhibition of p38γ and PFKFB3 relieved BLM-induced skin fibrosis in vivo. After injected with SB203580 and 3PO, p38γ and PFKFB3 expression in BLM-induced mice skin tissues was measured employing western blot ( A ). The dermal thickness and collagen thickness of mice skin tissues were detected utilizing H&E and Masson’s trichrome staining ( B ). Immunohistochemical staining for α-SMA and Ki67 in BLM-induced mice skin tissues ( C ). * P < 0.05, ** P < 0.01

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the primary antibodies in 5% milk-TBST overnight at 4 °C using the following concentrations: SPARC (1:500, no. ab290636 and ab225716, Abcam, UK), p38γ (1:1000, no. 20184–1-AP, Proteintech, USA), PFKFB3 (1:1000, no. 13763–1-AP, Proteintech, USA), α-SMA (1:1000, no. #19245, Cell Signaling Technology, USA), Fibronectin (1:500, no. ab2413, Abcam, UK), Collagen I (1:500, no. ab138492, Abcam, UK), Collagen III (1:500, no. ab184993, Abcam, UK) and β-actin (1:2000, no. ab8227, Abcam, UK).

Techniques: Inhibition, In Vivo, Injection, Expressing, Western Blot, Staining, Immunohistochemical staining

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: Verification of mRNA and protein levels of candidate senescence-specific genes in osteoarthritis. (A) The mRNA levels of MAPK12, FOS, CYR61, TNFSF15 were investigated in human normal control and osteoarthritis (OA) cartilage tissue by qRT-PCR, n = 6. (B) HE, Safranine O staining and protein levels of MAPK12, FOS, CYR61, TNFSF15 were investigated in human normal control and osteoarthritis (OA) cartilage tissue by immunohistochemistry, n = 6. Scale bar = 100 μm. (C) The mRNA levels of MAPK12, FOS, CYR61, TNFSF15 were investigated in human C28/I2 chondrocytes treated with 10 ng/ml IL-1β for 48h by qRT-PCR, n = 3. (D) The protein levels of MAPK12, FOS, CYR61, TNFSF15 were investigated in human C28/I2 chondrocytes treated with 10 ng/ml IL-1β for 48h by western blotting, n = 3. Statistical analysis was conducted using unpaired Student's t test. P values were compared between OA group and normal control group, between IL-1β group and PBS control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Knee joint of mice or human cartilage paraffin sections were digested using antigen repair solution (BOSTER, Cat. AR0026) at 37 °C for 30 min. 3 % hydrogen peroxide was incubated at room temperature for 10 min, followed by overnight incubation with corresponding primary antibody, Col2 (Proteintech, 28459-1-AP), ACAN (Affinity, DF7561), MMP13 (Proteintech, 18165-1-AP), ADAMTS5 (Affinity, DF13268), γ-H2AX (Cell Signaling Technology, 9718), IL6 (Affinity, DF6087), MAPK12 (Proteintech, 20184-1-AP), FOS (Proteintech, 66590-1-Ig), CYR61 (Proteintech, 67656-1-Ig) and TNFSF15 (Proteintech, 29899-1-AP) at 4 °C, and then immunoreactivity was detected using streptavidin-biotin assay system (ORIGENE, Cat. SP-9000).

Techniques: Control, Quantitative RT-PCR, Staining, Immunohistochemistry, Western Blot

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: MAPK12 over-expression inhibits senescence and DNA damage of C28/I2 cells induced by H 2 O 2 . (A) The protein levels of LMNB1, PCNA, p16, p21, MMP13 were investigated in C28/I2 cells by western blotting, n = 3. (B) EdU staining was adopted for cell proliferation capability of C28/I2 cells, n = 3. Scale bar = 100 μm. (C) The protein level of γ-H2AX was investigated in C28/I2 cells by immunofluorescence, n = 3. Scale bar = 100 μm. (D) SA-β-gal staining was adopted for senescence of C28/I2 cells, n = 3. Scale bar = 100 μm. Statistical analysis was conducted using one-way ANOVA. P values were compared between OE-MAPK12 group or control group and H 2 O 2 group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Knee joint of mice or human cartilage paraffin sections were digested using antigen repair solution (BOSTER, Cat. AR0026) at 37 °C for 30 min. 3 % hydrogen peroxide was incubated at room temperature for 10 min, followed by overnight incubation with corresponding primary antibody, Col2 (Proteintech, 28459-1-AP), ACAN (Affinity, DF7561), MMP13 (Proteintech, 18165-1-AP), ADAMTS5 (Affinity, DF13268), γ-H2AX (Cell Signaling Technology, 9718), IL6 (Affinity, DF6087), MAPK12 (Proteintech, 20184-1-AP), FOS (Proteintech, 66590-1-Ig), CYR61 (Proteintech, 67656-1-Ig) and TNFSF15 (Proteintech, 29899-1-AP) at 4 °C, and then immunoreactivity was detected using streptavidin-biotin assay system (ORIGENE, Cat. SP-9000).

Techniques: Over Expression, Western Blot, Staining, Immunofluorescence, Control

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: Over-expression of MAPK12 promotes cartilage anabolism and proliferation, inhibits cartilage catabolism and senescence of IL-1β stimulated C28/I2 cells. (A, B) The protein levels and quantification of LMNB1, PCNA, p16, p21, COL2, ACAN, SOX9, MMP13, ADAMTS5 were investigated in C28/I2 cells by western blotting, n = 3. (C, D) Representative image and quantification of EdU staining adopted for cell proliferation ability of C28/I2 cells, n = 3. Scale bar = 100 μm. Statistical analysis was conducted using one-way ANOVA. P values were compared between OE-MAPK12 group or control group and IL-1β group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Knee joint of mice or human cartilage paraffin sections were digested using antigen repair solution (BOSTER, Cat. AR0026) at 37 °C for 30 min. 3 % hydrogen peroxide was incubated at room temperature for 10 min, followed by overnight incubation with corresponding primary antibody, Col2 (Proteintech, 28459-1-AP), ACAN (Affinity, DF7561), MMP13 (Proteintech, 18165-1-AP), ADAMTS5 (Affinity, DF13268), γ-H2AX (Cell Signaling Technology, 9718), IL6 (Affinity, DF6087), MAPK12 (Proteintech, 20184-1-AP), FOS (Proteintech, 66590-1-Ig), CYR61 (Proteintech, 67656-1-Ig) and TNFSF15 (Proteintech, 29899-1-AP) at 4 °C, and then immunoreactivity was detected using streptavidin-biotin assay system (ORIGENE, Cat. SP-9000).

Techniques: Over Expression, Western Blot, Staining, Control

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: MAPK12 over-expression and FOS deficiency relieve osteoarthritis and joint pain in OA. (A) Representative images of HE, Safranine O staining and synovium in mice knee joint with or without MAPK12 over-expression or FOS knock-down adenovirus, n = 6. (B) OARSI score, meniscus total scores and synovitis score of mice knee joint with or without MAPK12 over-expression or FOS knock-down adenovirus, n = 6. (C, D) Representative images and quantification of Col2, ACAN, MMP13, Adamts5 protein levels in the knee cartilage of mice by immunohistochemistry, n = 6. Scale bar = 100 μm. (E) Paw withdrawl threshold of mice hind paw was measured using von frey filaments at 6 and 8 weeks post-DMM surgery, n = 6. (F) Response time of mice hind paw retraction or licking was recorded using hot plate at 6 and 8 weeks post-DMM surgery, n = 6. P values were compared between OE-MAPK12 and OE-control group, between siFOS group and si-control group, between OE-control or si-control group and sham group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Knee joint of mice or human cartilage paraffin sections were digested using antigen repair solution (BOSTER, Cat. AR0026) at 37 °C for 30 min. 3 % hydrogen peroxide was incubated at room temperature for 10 min, followed by overnight incubation with corresponding primary antibody, Col2 (Proteintech, 28459-1-AP), ACAN (Affinity, DF7561), MMP13 (Proteintech, 18165-1-AP), ADAMTS5 (Affinity, DF13268), γ-H2AX (Cell Signaling Technology, 9718), IL6 (Affinity, DF6087), MAPK12 (Proteintech, 20184-1-AP), FOS (Proteintech, 66590-1-Ig), CYR61 (Proteintech, 67656-1-Ig) and TNFSF15 (Proteintech, 29899-1-AP) at 4 °C, and then immunoreactivity was detected using streptavidin-biotin assay system (ORIGENE, Cat. SP-9000).

Techniques: Over Expression, Staining, Knockdown, Immunohistochemistry, Control

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: MAPK12 over-expression and FOS deficiency inhibit chondrocyte senescence in OA. (A) p16 protein levels were investigated in knee cartilage of mice by immunofluorescence. Scale bar = 50 μm. (B) p21 protein levels were investigated in knee cartilage of mice by immunofluorescence. Scale bar = 50 μm. (C) γ-H2AX protein levels were investigated in knee cartilage of mice by immunohistochemistry. Scale bar = 100 μm. (D) IL6 protein levels were investigated in knee cartilage of mice by immunohistochemistry. Scale bar = 100 μm. (E) Quantification of p16, p21, γ-H2AX and IL6 protein levels in knee cartilage of mice, n = 6. (F) Schematic illustration of the MAPK12 and FOS signaling pathways in OA by regulating cellular senescence. Statistical analysis was conducted using one-way ANOVA. P values were compared between OE-MAPK12 and OE-control group, between siFOS group and si-control group, between OE-control or si-control group and sham group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Knee joint of mice or human cartilage paraffin sections were digested using antigen repair solution (BOSTER, Cat. AR0026) at 37 °C for 30 min. 3 % hydrogen peroxide was incubated at room temperature for 10 min, followed by overnight incubation with corresponding primary antibody, Col2 (Proteintech, 28459-1-AP), ACAN (Affinity, DF7561), MMP13 (Proteintech, 18165-1-AP), ADAMTS5 (Affinity, DF13268), γ-H2AX (Cell Signaling Technology, 9718), IL6 (Affinity, DF6087), MAPK12 (Proteintech, 20184-1-AP), FOS (Proteintech, 66590-1-Ig), CYR61 (Proteintech, 67656-1-Ig) and TNFSF15 (Proteintech, 29899-1-AP) at 4 °C, and then immunoreactivity was detected using streptavidin-biotin assay system (ORIGENE, Cat. SP-9000).

Techniques: Over Expression, Immunofluorescence, Immunohistochemistry, Protein-Protein interactions, Control

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: Verification of mRNA and protein levels of candidate senescence-specific genes in osteoarthritis. (A) The mRNA levels of MAPK12, FOS, CYR61, TNFSF15 were investigated in human normal control and osteoarthritis (OA) cartilage tissue by qRT-PCR, n = 6. (B) HE, Safranine O staining and protein levels of MAPK12, FOS, CYR61, TNFSF15 were investigated in human normal control and osteoarthritis (OA) cartilage tissue by immunohistochemistry, n = 6. Scale bar = 100 μm. (C) The mRNA levels of MAPK12, FOS, CYR61, TNFSF15 were investigated in human C28/I2 chondrocytes treated with 10 ng/ml IL-1β for 48h by qRT-PCR, n = 3. (D) The protein levels of MAPK12, FOS, CYR61, TNFSF15 were investigated in human C28/I2 chondrocytes treated with 10 ng/ml IL-1β for 48h by western blotting, n = 3. Statistical analysis was conducted using unpaired Student's t test. P values were compared between OA group and normal control group, between IL-1β group and PBS control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Cells were treated with 10 ng/ml IL-1β or 400 μM H 2 O 2 to establish OA or senescence model in vitro , then transfected with siRNA (siFOS forward: 5′-ACCUAUCUGGGUCCUUCUAUGtt-3′, siFOS reverse: 5′-CAUAGAAGGACCCAGAUAGGUtt-3′, synthesized by Tsingke Biotechnology) or pcDNA3.1-MAPK12 plasmid by Lipofectamine 2000 (ThermoFisher, 11668030) for 48 h.

Techniques: Control, Quantitative RT-PCR, Staining, Immunohistochemistry, Western Blot

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: MAPK12 over-expression inhibits senescence and DNA damage of C28/I2 cells induced by H 2 O 2 . (A) The protein levels of LMNB1, PCNA, p16, p21, MMP13 were investigated in C28/I2 cells by western blotting, n = 3. (B) EdU staining was adopted for cell proliferation capability of C28/I2 cells, n = 3. Scale bar = 100 μm. (C) The protein level of γ-H2AX was investigated in C28/I2 cells by immunofluorescence, n = 3. Scale bar = 100 μm. (D) SA-β-gal staining was adopted for senescence of C28/I2 cells, n = 3. Scale bar = 100 μm. Statistical analysis was conducted using one-way ANOVA. P values were compared between OE-MAPK12 group or control group and H 2 O 2 group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Cells were treated with 10 ng/ml IL-1β or 400 μM H 2 O 2 to establish OA or senescence model in vitro , then transfected with siRNA (siFOS forward: 5′-ACCUAUCUGGGUCCUUCUAUGtt-3′, siFOS reverse: 5′-CAUAGAAGGACCCAGAUAGGUtt-3′, synthesized by Tsingke Biotechnology) or pcDNA3.1-MAPK12 plasmid by Lipofectamine 2000 (ThermoFisher, 11668030) for 48 h.

Techniques: Over Expression, Western Blot, Staining, Immunofluorescence, Control

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: Over-expression of MAPK12 promotes cartilage anabolism and proliferation, inhibits cartilage catabolism and senescence of IL-1β stimulated C28/I2 cells. (A, B) The protein levels and quantification of LMNB1, PCNA, p16, p21, COL2, ACAN, SOX9, MMP13, ADAMTS5 were investigated in C28/I2 cells by western blotting, n = 3. (C, D) Representative image and quantification of EdU staining adopted for cell proliferation ability of C28/I2 cells, n = 3. Scale bar = 100 μm. Statistical analysis was conducted using one-way ANOVA. P values were compared between OE-MAPK12 group or control group and IL-1β group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Cells were treated with 10 ng/ml IL-1β or 400 μM H 2 O 2 to establish OA or senescence model in vitro , then transfected with siRNA (siFOS forward: 5′-ACCUAUCUGGGUCCUUCUAUGtt-3′, siFOS reverse: 5′-CAUAGAAGGACCCAGAUAGGUtt-3′, synthesized by Tsingke Biotechnology) or pcDNA3.1-MAPK12 plasmid by Lipofectamine 2000 (ThermoFisher, 11668030) for 48 h.

Techniques: Over Expression, Western Blot, Staining, Control

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: MAPK12 over-expression and FOS deficiency relieve osteoarthritis and joint pain in OA. (A) Representative images of HE, Safranine O staining and synovium in mice knee joint with or without MAPK12 over-expression or FOS knock-down adenovirus, n = 6. (B) OARSI score, meniscus total scores and synovitis score of mice knee joint with or without MAPK12 over-expression or FOS knock-down adenovirus, n = 6. (C, D) Representative images and quantification of Col2, ACAN, MMP13, Adamts5 protein levels in the knee cartilage of mice by immunohistochemistry, n = 6. Scale bar = 100 μm. (E) Paw withdrawl threshold of mice hind paw was measured using von frey filaments at 6 and 8 weeks post-DMM surgery, n = 6. (F) Response time of mice hind paw retraction or licking was recorded using hot plate at 6 and 8 weeks post-DMM surgery, n = 6. P values were compared between OE-MAPK12 and OE-control group, between siFOS group and si-control group, between OE-control or si-control group and sham group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Cells were treated with 10 ng/ml IL-1β or 400 μM H 2 O 2 to establish OA or senescence model in vitro , then transfected with siRNA (siFOS forward: 5′-ACCUAUCUGGGUCCUUCUAUGtt-3′, siFOS reverse: 5′-CAUAGAAGGACCCAGAUAGGUtt-3′, synthesized by Tsingke Biotechnology) or pcDNA3.1-MAPK12 plasmid by Lipofectamine 2000 (ThermoFisher, 11668030) for 48 h.

Techniques: Over Expression, Staining, Knockdown, Immunohistochemistry, Control

Journal: Journal of Orthopaedic Translation

Article Title: Targeting the senescence-related genes MAPK12 and FOS to alleviate osteoarthritis

doi: 10.1016/j.jot.2024.06.008

Figure Lengend Snippet: MAPK12 over-expression and FOS deficiency inhibit chondrocyte senescence in OA. (A) p16 protein levels were investigated in knee cartilage of mice by immunofluorescence. Scale bar = 50 μm. (B) p21 protein levels were investigated in knee cartilage of mice by immunofluorescence. Scale bar = 50 μm. (C) γ-H2AX protein levels were investigated in knee cartilage of mice by immunohistochemistry. Scale bar = 100 μm. (D) IL6 protein levels were investigated in knee cartilage of mice by immunohistochemistry. Scale bar = 100 μm. (E) Quantification of p16, p21, γ-H2AX and IL6 protein levels in knee cartilage of mice, n = 6. (F) Schematic illustration of the MAPK12 and FOS signaling pathways in OA by regulating cellular senescence. Statistical analysis was conducted using one-way ANOVA. P values were compared between OE-MAPK12 and OE-control group, between siFOS group and si-control group, between OE-control or si-control group and sham group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: Cells were treated with 10 ng/ml IL-1β or 400 μM H 2 O 2 to establish OA or senescence model in vitro , then transfected with siRNA (siFOS forward: 5′-ACCUAUCUGGGUCCUUCUAUGtt-3′, siFOS reverse: 5′-CAUAGAAGGACCCAGAUAGGUtt-3′, synthesized by Tsingke Biotechnology) or pcDNA3.1-MAPK12 plasmid by Lipofectamine 2000 (ThermoFisher, 11668030) for 48 h.

Techniques: Over Expression, Immunofluorescence, Immunohistochemistry, Control