Journal: Translational Oncology

Article Title: Can thymosin beta 10 function both as a non-invasive biomarker and chemotherapeutic target in human colorectal cancer?

doi: 10.1016/j.tranon.2024.102026

Figure Lengend Snippet: Knockdown of TMSB10 alters p38 pathways in human CRC. (A) TMSB10, p38, p-p38, p53, p-p53, caspase 3, p21, Rb and p-Rb were examined in both TMSB10-knockdown cells. (B) Ratio of p-p38/p38. (C) Ratio of p-p53/p53. (D) Ratio of p-Rb/Rb. (E) The correlation analysis between TMSB10 and p-p38 was carried out by Pearson correlation coefficient. (F and G) The expressions of TMSB10, Ki67, p-p38, p-p53, caspase 3, p21, p-Rb expression in tumor from mice injected with TMSB10-knockdown cells and negative control cells was were examined by IHC. All data are expressed as mean ± standard deviation (SD; three independent experiments), and statistical significance is denoted by * between different groups ( p < 0.05).

Article Snippet: After washing with PBST, the separated proteins were incubated with the special antibodies including anti-p38 (14,064–1-AP, ProteinTech, Chicago, USA), anti-p38 (phosphoT180, ab178867, Abcam, Cambridge, UK), anti-p53 (10,442–1-AP, ProteinTech, Chicago, USA), anti-p53 (phospho S15, ab1431, Abcam, Cambridge, UK), anti-p21 (ab109520, ProteinTech, Chicago, USA), anti-Rb (17,218–1-AP, ProteinTech, Chicago, USA), anti-Rb (phospho-S807, 8180T, Cell Signaling Technology, Frankfurt, Germany), and anti-Caspase3 (ab13847, Abcam, Cambridge, UK) at 4 °C overnight.

Techniques: Knockdown, Expressing, Injection, Negative Control, Standard Deviation

Journal: Translational Oncology

Article Title: Can thymosin beta 10 function both as a non-invasive biomarker and chemotherapeutic target in human colorectal cancer?

doi: 10.1016/j.tranon.2024.102026

Figure Lengend Snippet: Regulation model of TMSB10 on cell proliferation in CRC. Knockdown- TMSB10 promotes the phosphorylation of p38 and p53. The up-regulated p-p53 inhibits cell cycle progression and promotes apoptosis via the p21/Rb pathway and caspase3 pathway, respectively. Arrows (→) and truncated lines (—|) indicate promoting and inhibiting effects, respectively.

Article Snippet: After washing with PBST, the separated proteins were incubated with the special antibodies including anti-p38 (14,064–1-AP, ProteinTech, Chicago, USA), anti-p38 (phosphoT180, ab178867, Abcam, Cambridge, UK), anti-p53 (10,442–1-AP, ProteinTech, Chicago, USA), anti-p53 (phospho S15, ab1431, Abcam, Cambridge, UK), anti-p21 (ab109520, ProteinTech, Chicago, USA), anti-Rb (17,218–1-AP, ProteinTech, Chicago, USA), anti-Rb (phospho-S807, 8180T, Cell Signaling Technology, Frankfurt, Germany), and anti-Caspase3 (ab13847, Abcam, Cambridge, UK) at 4 °C overnight.

Techniques: Knockdown, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: Oxidative stress increases OGT and OGA expression, OGA activity, and O-GlcNAc levels. U2OS cells were treated with vehicle (V) or H2O2 (2.5 mm, 1–3 h). n = 10, unless otherwise indicated. A, expression of OGT, OGA, and actin, as well as O-GlcNAc levels, was assessed in NETN lysates (5 μg) by Western blotting (WB). Protein load was assessed by total protein stain (colloidal Coomassie G-250) and by Western blotting (actin). Molecular mass (MW) markers are indicated. B, quantitation of actin normalized to total protein (G-250). C, quantitation of O-GlcNAc levels normalized to actin. D, quantitation of OGT expression normalized to actin. E, NETN lysates (5 μg) were assayed for OGT activity using [3H]UDP-GlcNAc (0.5 μCi) and CKII acceptor peptide (1 mm). n = 4, three technical replicates per assay. F, quantitation of OGA expression normalized to actin. G, NETN lysates (5 μg) were assayed for OGA activity using 4MU-GlcNAc (1 mm). n = 6, two technical replicates per assay. B–G, data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Dunnett's MCT, and differences were considered statistically significant at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.0001 (****).

Article Snippet: In a clear round-bottomed 96-well plate, 5 μg of cell lysate was incubated (1 h, 25 °C) in triplicate with 0.5 μCi of [ 3 H]UDP-GlcNAc (ART 0128; American Radiolabeled Chemicals, St. Louis, MO; specific activity, 60 Ci/mmol), 1 m m casein kinase II (CKII) acceptor peptide (PGGSTPVSSANMM; The Johns Hopkins University School of Medicine Synthesis and Sequencing Facility), 2.5 units of calf intestinal alkaline phosphatase (New England Biolabs), 0.25 m m 5′-AMP (Sigma), and OGT assay buffer (100 m m sodium cacodylate, pH 6.4, 0.3% (w/v) BSA).

Techniques: Expressing, Activity Assay, Western Blot, Staining, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: Oxidative stress increases OGT and OGA expression, OGA activity, and O-GlcNAc levels. U2OS cells were treated with vehicle (V) or H2O2 (2.5 mm, 1–3 h). n = 10, unless otherwise indicated. A, expression of OGT, OGA, and actin, as well as O-GlcNAc levels, was assessed in NETN lysates (5 μg) by Western blotting (WB). Protein load was assessed by total protein stain (colloidal Coomassie G-250) and by Western blotting (actin). Molecular mass (MW) markers are indicated. B, quantitation of actin normalized to total protein (G-250). C, quantitation of O-GlcNAc levels normalized to actin. D, quantitation of OGT expression normalized to actin. E, NETN lysates (5 μg) were assayed for OGT activity using [3H]UDP-GlcNAc (0.5 μCi) and CKII acceptor peptide (1 mm). n = 4, three technical replicates per assay. F, quantitation of OGA expression normalized to actin. G, NETN lysates (5 μg) were assayed for OGA activity using 4MU-GlcNAc (1 mm). n = 6, two technical replicates per assay. B–G, data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Dunnett's MCT, and differences were considered statistically significant at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.0001 (****).

Article Snippet: In a clear round-bottomed 96-well plate, 5 μg of cell lysate was incubated (1 h, 25 °C) in triplicate with 0.5 μCi of [ 3 H]UDP-GlcNAc (ART 0128; American Radiolabeled Chemicals, St. Louis, MO; specific activity, 60 Ci/mmol), 1 m m casein kinase II (CKII) acceptor peptide (PGGSTPVSSANMM; The Johns Hopkins University School of Medicine Synthesis and Sequencing Facility), 2.5 units of calf intestinal alkaline phosphatase (New England Biolabs), 0.25 m m 5′-AMP (Sigma), and OGT assay buffer (100 m m sodium cacodylate, pH 6.4, 0.3% (w/v) BSA).

Techniques: Expressing, Activity Assay, Western Blot, Staining, Quantitation Assay