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: Advanced Science

Article Title: Illuminating Mitochondrial RNA G‐Quadruplexes as Structural Brakes on RNA Granule Assembly and OXPHOS

doi: 10.1002/advs.202523462

Figure Lengend Snippet: A chemical genetic screen identifies the Wnt/β‐Catenin‐GRSF1‐mtRNA G4 axis regulating MRG assembly in cancer cells. (A) Depiction of the screening workflow. The data obtained regarding the fluorescence changes of MitoQUMA and MitoTO were standardized by calculating the Z‐ score for each inhibitor. (B) Z ‐score value for each inhibitor was plotted to identify hits with thresholds of | Z ‐score ( MitoQUMA ) |>3 and |Z‐score (MitoTO) | <0.5. (C) Live HeLa cells were treated with varying concentrations of ICG‐001, followed by staining with 2 µ m MitoQUMA . (D) Live HeLa cells were transfected with siRNA to knock down GRSF1 expression and then treated with or without 20 n m BML‐284, followed by staining with 2 µ m MitoQUMA . (E) Influence of MRG formation in HeLa cells upon siGRSF1 treatment with or without subsequent 20 nM BML‐284 exposure. MRGs were indicated by immunofluorescence staining of FASTKD2. For each sample of cell image, approximately 100 cells were measured. Biological replicates ( n = 3) were taken. The data are presented as mean ± SEM, and statistical significance is determined by the one‐way ANOVA with Dunnett's multiple comparisons test as (ns) not significant, ( * ) p < 0.05, ( ** ) p < 0.01, and ( *** ) p < 0.001. Scale bars for cell image: 10 µm.

Article Snippet: The treatment concentrations of IMT1 (TargetMol, T8841), Chloramphenicol (TargetMol, T1205), RHPS4 (TargetMol, T6967), Valinomycin (TargetMol, TP1072), Ionomycin (Beyotime, S1672), ICG‐001 (TargetMol, T6113) and BML‐284 (TargetMol, T8820) have been provided in the figure legend.

Techniques: Screening, Fluorescence, Staining, Transfection, Knockdown, Expressing, Immunofluorescence

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