Journal: Cell Death & Disease

Article Title: Oncogene PRR14 promotes breast cancer through activation of PI3K signal pathway and inhibition of CHEK2 pathway

doi: 10.1038/s41419-020-2640-8

Figure Lengend Snippet: CHEK2 mRNA expression ( a ), CHEK2 protein expression ( b ) and p-CHEK2 (T68) protein expression ( c ) in PRR14 genetically unaltered and altered (amplified and mutated) breast cancer cases in TCGA database are statistically analyzed by two-tailed Student’s t -test. CHEK2 protein expression is detected by immunostaining in xenografts in nude mice from established MCF7 cell lines and MDA-MB-231 cell lines ( d ), as well as human breast cancer ( e ). CHEK2 transcription in human breast cancer is also detected by qRT-PCR ( f ). The data are quantified and two-tailed Student’s t -test is employed to determine the significance of the difference. Established MCF7 and MDA-MB-231 ( g ) PRR14-overexpressing and control cell lines are treated with various of genotoxic chemicals including Bleo, Eto, 5-FU, H2O2 and HU at indicated concentrations for indicated time. Key components of the ATM/CHEK2/P53 signaling pathway are detected by immunostaining. And CHEK2 protein expression ( h ) and mRNA expression ( i ) are detected by immunostaining and qRT-PCR, respectively. The data are analyzed by two-tailed Student’s t -test. Established MCF7 and MDA-MB-231 ( j ) PRR14-overexpressing and control cell lines are treated with Eto at indicated concentration for indicated time to induce p-CHEK2 (T68), which is detected by immunostaining and quantified and normalized by CHEK2 total protein ( k ). The data are analyzed by two-tailed Student’s t -test.

Article Snippet: Chemicals including selective CHEK2 inhibitor BML-277 (BML, HY-13946, MCE) , and genotoxic chemicals, including bleomycin (Bleo, HY-17565, MCE), etoposide (Eto, HY-13629, MCE), 5-fluorouracil (5-FU, HY-90006, MCE), H 2 O 2 (88597, Millipore) and hydroxyurea (HU, HY-B0313, MCE), were used to treat cells.

Techniques: Expressing, Amplification, Two Tailed Test, Immunostaining, Quantitative RT-PCR, Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: Oncogene PRR14 promotes breast cancer through activation of PI3K signal pathway and inhibition of CHEK2 pathway

doi: 10.1038/s41419-020-2640-8

Figure Lengend Snippet: MCF7 cells transfected with indicated siRNA sequences for 48 h are treated w/wo 5 μg/ml Eto for 6 h, then cells are harvested for immunostaining ( a ). PRR14 in cells is depleted by RNAi transfection for 48 h, and CHEK2 mRNA level are quantified by qRT-PCR in both MCF7 and MDA-MB-231 cell lines ( b ). MCF7, 10AKRAS, 7E6 and MDA-MB-231 cell lines are treated with increasing concentration of CHEK2 inhibitor BML for 24 h and followed with a 24 h treatment of Eto at 5 μg/ml. Cells are harvested to stain with PI followed with FACS analysis ( c ). The percentage of the 4N fraction is analyzed by one-way ANOVA analysis ( n = 3) ( d ).

Article Snippet: Chemicals including selective CHEK2 inhibitor BML-277 (BML, HY-13946, MCE) , and genotoxic chemicals, including bleomycin (Bleo, HY-17565, MCE), etoposide (Eto, HY-13629, MCE), 5-fluorouracil (5-FU, HY-90006, MCE), H 2 O 2 (88597, Millipore) and hydroxyurea (HU, HY-B0313, MCE), were used to treat cells.

Techniques: Transfection, Immunostaining, Quantitative RT-PCR, Concentration Assay, Staining

Journal: Cell Death & Disease

Article Title: Oncogene PRR14 promotes breast cancer through activation of PI3K signal pathway and inhibition of CHEK2 pathway

doi: 10.1038/s41419-020-2640-8

Figure Lengend Snippet: KM survival curves of breast cancer patients receiving chemotherapy are stratified by their expression levels of either CHEK2 ( a ) or PRR14 ( b ). For comparison, KM survival curves of breast cancer patients receiving endocrine therapy and stratified by PRR14 expression is also analyzed ( c ). KM survival curves of breast cancer patients receiving chemotherapy with mutant P53 ( d ) or wild-type P53 ( e ) are stratified by their expression level of PRR14.

Article Snippet: Chemicals including selective CHEK2 inhibitor BML-277 (BML, HY-13946, MCE) , and genotoxic chemicals, including bleomycin (Bleo, HY-17565, MCE), etoposide (Eto, HY-13629, MCE), 5-fluorouracil (5-FU, HY-90006, MCE), H 2 O 2 (88597, Millipore) and hydroxyurea (HU, HY-B0313, MCE), were used to treat cells.

Techniques: Expressing, Comparison, Mutagenesis

Journal: Cancer Cell International

Article Title: Sestrin2 reduces cancer stemness via Wnt/β-catenin signaling in colorectal cancer

doi: 10.1186/s12935-022-02498-x

Figure Lengend Snippet: The effects of Sestrin2 on cancer stemness are mediated by the Wnt/β-catenin pathway. A Western blot analysis of Wnt/β-catenin signaling-related proteins. The relative protein expression is shown in the right panel. β-Actin was used as a loading control * P = 0.05; Mann–Whitney test; lines showed medians). B Sphersphere formation assay in LV-Sestrin2 and LV-GFP CRC cells with or without exposure to BML-284 (0.1 µM). C Number of spheres, * compared with LV-GFP, * P = 0.05; # compared with LV-Sestrin2, # P = 0.05; Mann–Whitney test; lines showed medians. D Western blot analysis of proteins related to the Wnt/β-catenin pathway (β-catenin, c-Myc) and cancer stemness (CD44) in LV-Sestrin2 and LV-GFP CRC cells with or without incubation with BML-284 (0.5 µM) for 24 h in HCT-116 cells. E The relative protein expression. * Compared with LV-GFP, * P = 0.05, # compared with LV-Sestrin2, # P = 0.05, Mann–Whitney test; lines showed medians

Article Snippet: HCT-116 cells in the LV-Sestrin2 and LV-GFP groups were treated with 0.5 μM BML-284 (Wnt signaling activator; MedChemExpress, Monmouth Junction, NJ, USA) in dimethyl sulfoxide for 24 h and then collected for western blot analysis.

Techniques: Western Blot, Expressing, Control, MANN-WHITNEY, Tube Formation Assay, Incubation

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Drp1 acetylation mediated by CDK5-AMPK-GCN5L1 axis promotes cerebral ischemic injury via facilitating mitochondrial fission.

doi: 10.1186/s10020-024-00948-y

Figure Lengend Snippet: Fig. 8 Inhibition of AMPK by Compound C protects brain tissues from ischemic damage in mouse models of dMCAO. (A) Representative TTC-stained brain sections and quantitative analysis of infarct volume (n=3). (B) Western blot analysis of p-Drp1 and p-AMPK in brain tissues of dMCAO and control mice treated with or without Compound C (n=3). (C) Immunohistochemistry staining for p-Drp1 and p-AMPK in brain tissues of dMCAO and control mice treated as in (B). Scale bars represent 100 μm (n=3). (D) Acetylated Drp1 was analyzed in brain tissues of dMCAO and control mice treated as in (B) by IP with anti-acetyl lysine antibody and western blotting with anti-Drp1 (n=3). (E) Fluorescence images of MitoSOX staining in brain sections of dMCAO and control mice treated as in (B). Scale bars represent 500 μm (n=3). (F) mtROS levels were analyzed by flow cytometry. Bar graphs show the mtROS levels measured based on fluorescence intensity (n=3). (G) TUNEL staining detected apoptosis in brain tissues of dMCAO and control mice treated as in (B). Scale bars represent 200 μm (n=3). Data are represented as mean ± SD, *P<0.05, **P<0.01, ****P<0.0001, P-value was determined by one-way ANOVA with Dunnett’s post hoc correction

Article Snippet: Compound C (inhibitor of AMPK) (5 μM, MCE, HY-13418 A) was acquired from MCE and dissolved in DMSO.

Techniques: Inhibition, Staining, Western Blot, Control, Immunohistochemistry, Fluorescence, Flow Cytometry, TUNEL Assay

Journal: Renal Failure

Article Title: Hypoxia-inducible factor 2α overexpression in podocytes ameliorates lipid metabolism disorders in diabetic kidney disease by inhibiting S1P

doi: 10.1080/0886022X.2025.2568972

Figure Lengend Snippet: HIF-2α regulated lipid metabolism in podocytes via S1P-S1PR3. (A). The mRNA level of genes involved in cholesterol synthesis, uptake, and efflux in podocytes with different treatments ( n = 3). (B). Representative western blotting and quantification of ABCA1 and HIF-2α in podocytes with different treatments ( n = 3). (C). The mRNA level of S1PR-family members in podocytes ( n = 3). (D). Representative western blotting and quantification of S1PR1, S1PR2, and S1PR3 in podocytes ( n = 3). (E). Representative western blotting and quantification of S1PR3 in podocytes stimulated by S1P and S1PR3 inhibitors ( n = 3). (F). The levels of total cholesterol in podocytes with different treatments ( n = 3). (G). The mRNA level of ABCA1 in podocytes stimulated by S1P and S1PR3 inhibitors ( n = 3). (H). Representative western blotting and quantification of ABCA1, ERK1/2, and p-ERK1/2 in podocytes ( n = 3). NG: normal glucose; NG+MA: normal glucose with 24.4 mmol/L mannitol; HG: high glucose; HG+OE-NC: high glucose with OE-NC; HG+OE-HIF-2α: high glucose with OE-HIF-2α; HG+S1P: high glucose with 0.5 μmol/L S1P; HG+S1P + CAY10444: high glucose with 0.5 μmol/L S1P and 10 μmol/L CAY10444. Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were then exposed to different stimuli for 72 h in the following manner: (1) normal glucose (NG): the final concentration of glucose in the medium was 5.6 mmol/L; (2) normal glucose + mannitol (NG + MA): medium containing 5.6 mmol/L glucose was supplemented with 24.4 mmol/L of mannitol as an osmolality control for high glucose; (3) high glucose (HG): the final concentration of glucose in the medium was 30 mmol/L; (4) high glucose + 0.5 μmol/L of S1P (MedChemExpress, New Jersey, USA, HG + S1P); (5) high glucose + 0.5 μmol/L of S1P + 10 μmol/L of CAY10444 (S1PR3 inhibitor, MedChemExpress, New Jersey, USA, HG + S1P + CAY10444); (6) high glucose + 1 μmol/L of PF-543 (SPHK1 inhibitor, MedChemExpress, New Jersey, USA, HG + PF-543); (7) high glucose + 50 μmol/L FG-4592 (HIF prolyl hydroxylase inhibitor, Selleck, Texas, USA, HG + FG-4592).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Fast-Acting and Receptor-Mediated Regulation of Neuronal Signaling Pathways by Copaiba Essential Oil

doi: 10.3390/ijms21072259

Figure Lengend Snippet: CB2 agonists abrogated the activation of the PI3K/Akt/mTOR signaling pathway by copaiba essential oil. ( A ) Pan-Akt profiles and ( B ) expression levels of the mTOR and p70S6K phosphoisoforms in SH-SY5Y cells at 30 min following treatment with copaiba essential oil (100 ng/mL), AM1241 (200 nM), BML190 (10 µM), 100 ng/mL copaiba essential oil together with 200 nM AM1241 (CPB/AM), or 100 ng/mL copaiba essential oil together with 10 µM BML190 (CPB/BML). ( C ) Relative concentrations of the Akt1 (blue), Akt2 (orange), Akt3 (gray), mTOR (yellow), and p70S6K (purple) phosphoisoforms in SH-SY5Y cells at 30 min following treatment with copaiba essential oil, AM1241, BML190, CPB/AM, or CPB/BML. The relative concentration describes the fold change in a protein phosphoisoform after treatment compared to the control condition. The error bars are the standard deviations of six repeated measurements per experimental condition. The asterisks indicate statistical significance for p ≤ 0.05 versus the control.

Article Snippet: The culture medium was replaced with culture media premixed with copaiba essential oil (100 ng/mL) or serial dilutions of copaiba essential oil, the CB2 agonist AM1241 (200 nM, Selleckchem, Houston, TX, USA), the CB2 inverse agonist BML190 (10 μM, Selleckchem), copaiba essential oil (100 ng/mL) and AM1241 (200 nM), or copaiba essential oil (100 ng/mL) and BML190 (10 μM).

Techniques: Activation Assay, Expressing, Concentration Assay, Control

Journal: Frontiers in Pharmacology

Article Title: S1P/S1PR1 signaling is involved in the development of nociceptive pain

doi: 10.3389/fphar.2024.1407347

Figure Lengend Snippet: SPHK1 inhibitor reduced nociceptive pain. (A) The content of S1P in the serum, DRG and spinal cord of CFA mice on day 3 was analyzed by ELISA after administration of SK1-I (5 mg/kg, 10 mg/kg) (n = 3 biological repeats, 3 mice/repeat/group). Data are shown as the mean ± SEM. * p < 0.05 vs. Vehicle. (B–D) The PWF and PWL of CFA mice after intraperitoneal administration of the SPHK1 inhibitor SK1-I (n = 6 mice/group). Data are shown as the mean ± SEM. * p < 0.05, *** p < 0.001 vs. CFA + Vehicle. (E) The activation of astrocytes and microglial cells in the spinal dorsal horn was detected by immunofluorescence after SK1-I injection (n = 3–4 biological repeats). (F,G) The immunoreactivity levels of GFAP and IBA1 in the spinal dorsal horn after SK1-I administration (n = 3–4 biological repeats). Data are shown as the mean ± SEM. * p < 0.1 vs. CFA + Vehicle. The red arrow represents the time of model establishment. The green arrow represents the time of drug administration.

Article Snippet: Complete Freund’s adjuvant (F5881) was purchased from Sigma‒Aldrich Canada Ltd. S1P (HY-108496, 5 μL, 100 Um), SK1-I (HY-119016), FTY720 (HY-12005), siponimod (HY-12355), and KRP-203 (HY-13660) were purchased from MCE ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Immunofluorescence, Injection