Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

doi: 10.1093/toxsci/kfac131

Figure Lengend Snippet: (A) A heatmap shows concentration-dependent changes of representative DNA repair-related genes. (B-E) Relative expression of EXO1 (B), MRE11 (C), NEIL3 (D), and FANCB (E) was measured using real-time PCR in CBD-treated human Sertoli cells. The bar graphs show means ± SD (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

Article Snippet: To measure gene expression levels, real-time PCR assays were performed using FastStart Universal Probe Master (Rox, MilliporeSigma) and the following probes (Thermo Fisher Scientific): E2F1 (Hs00153451_m1), E2F7 (Hs00987777_m1), MCM3 (Hs00172459_m1), PRIM1 (Hs00265388_m1), POLE2 (Hs00160277_m1), POLD1 (Hs01100821_m1), CDC25A (Hs00947994_m1), CDKN1A (Hs00355782_m1), EXO1 (Hs01116190_m1), MRE11 (Hs00967437_m1), NEIL3 (Hs00217387_m1), FANCB (Hs00537483_m1), CDKN2A (Hs00923894_m1), IL-1A (Hs00174092_m1), IL-6 (Hs00174131_m1), and GAPDH (Hs02758991_g1).

Techniques: Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Nature

Article Title: Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection.

doi: 10.1038/s41586-024-07909-9

Figure Lengend Snippet: Fig. 5 | BRCA1–BARD1 promotes resection by EXO1 independently of CtIP. a, Representative kinetic resection assays with EXO1, RPA, in the absence or presence of BRCA1–BARD1. Top, a schematic of the assay. Red asterisks (*) represent the position of the radioactive labels. b, Quantitation of DNA degradation from assays such as shown in a. Averages shown; error bars, s.e.m.; n = 3. c, Cartoon of the primary structure of the EXO1 protein and the Δ1 variant used in the study. d, Resection assays with RPA, wild-type EXO1 or EXO1 Δ1, in the absence or presence of BRCA1–BARD1. Top, quantitation of DNA degradation. Averages shown; error bars, s.e.m.; n = 3. Bottom, representative assays. e, Representative protein-interaction assays. Top, a schematic of the assay. f, Quantitation of clonogenic cell survival of RPE1 cells EXO1+/+ or EXO1−/−

Article Snippet: To test the interaction between BRCA1–BARD1 and WRN or EXO1, 1 μg of anti-BRCA1 antibody (Santa Cruz Biotechnology, sc-6954) or anti-WRN antibody (Cell Signaling, 4666S) were captured on 10 μl Protein G magnetic beads (Dynabeads, Invitrogen) by incubating at 4 °C for 1 h with gentle rotation in 50 μl of PBS-T (PBS with 0.1% Tween-20, Sigma).

Techniques: Quantitation Assay, Variant Assay

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: BRCA1/BCLAF1 Forms an mRNA Splicing Complex which Is Recruited to Target Gene Promoters and Transcripts following DNA Damage (A) Coimmunoprecipitation assays demonstrating that BCLAF1 interacts with the spliceosome proteins Prp8, U2AF65, U2AF35, and SF3B1 in both the presence and absence of DNA damage. (B) Coimmunoprecipitation assays demonstrating DNA damage-induced interaction between BRCA1 and the spliceosome proteins Prp8, U2AF65 U2AF35, and SF3B1 in response to DNA damage. Additionally, depletion of BCLAF1 results in abrogation of DNA damage-induced interaction between BRCA1 and these proteins. (C) BRCA1, BCLAF1, and U2AF65 ChIP-qPCRs demonstrating constitutive binding of BRCA1 to ATRIP , BACH1 , and EXO1 promoters irrespective of DNA damage in control (siCtrl) cells. The ChIPs also demonstrate that BCLAF1 and U2AF65 are recruited to these promoters only in etoposide-treated cells and that depletion of BRCA1 or BCLAF1 results in loss of DNA damage-induced BCLAF1 and U2AF65 recruitment, respectively. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM. (D) BRCA1, BCLAF1, and U2AF65 RIP-qRT-PCRs demonstrating that BRCA1, BCLAF1, and U2AF65 only bind to ATRIP , BACH1 , and EXO1 mRNAs in response to DNA damage. In addition, depletion of BCLAF1 results in loss of BRCA1 and U2AF65 mRNA binding to all three transcripts. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM. See also Figure S3 .

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Binding Assay, Control

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: The BRCA1/BCLAF mRNA Splicing Complex Promotes the Splicing and Stability of ATRIP , BACH1 , and EXO1 Transcripts following DNA Damage (A) Ratio of postspliced to prespliced ATRIP , BACH1 , and EXO1 mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells mock treated or treated with etoposide. mRNA levels were assessed by qRT-PCR using exon 9-exon 10 (post-spliced- ATRIP ) and exon 9-intron 9 (pre-spliced- ATRIP ), exon 15-exon 16 (post-spliced- BACH1 ) and exon 15-intron 15 (pre-spliced- BACH1 ), and exon 1-exon 2 (post-spliced- EXO1 ) and exon 1-intron 1 (pre-spliced- EXO1 ) primers and normalized to ACTB mRNA. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM. Significance of changes in splicing ratios was assessed using Student’s two-tailed t test with significant changes indicated by ∗∗ p < 0.01. (B) Semiquantitative PCR analysis of a cDNA generated from DNase-treated RNA, collected from control (siCtrl) and BRCA1- or BCLAF1-depleted cells, mock treated or treated with Etoposide. Primers targeting two independent intronic regions within ATRIP , BACH1 , and EXO1 and a single intronic region within ATM and CHEK2 (control genes) were used for semiquantitative PCR analysis. Exon-spanning primers targeted against ACTB were used as a loading control. (C) Normalized expression of prespliced and postspliced mRNAs evaluated in (A). (D) Expression levels of postspliced and prespliced ATRIP mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells, transfected with control siRNA (siCtrl) or depleted of SMG1 (siSMG1), a key regulator of the non-sense-mediated decay pathway. Normalized expression levels were quantified as in (A). Graphs represent the mean normalized expression from three independent experiments ± SEM. See also and .

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Control, Quantitative RT-PCR, Two Tailed Test, Generated, Expressing, Transfection

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: BRCA1 Ser-1423 Phosphorylation Is Required for BCLAF1 Recruitment and Target Gene Splicing following DNA Damage (A) BRCA1, pS1423-BRCA1, and BCLAF1 ChIP-qPCRs demonstrating constitutive binding of BRCA1 to ATRIP , BACH1 , and EXO1 promoters irrespective of DNA damage and DNA damage-dependent enrichment of pS1423-BRCA1 and BCLAF1 on these promoters. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM. (B–D) Ratio of postspliced to prespliced ATRIP , BACH1 , and EXO1 mRNA in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt-BRCA1), or S1423A Flag-BRCA1 (S1423A-BRCA1). Cells were mock treated or treated with etoposide. mRNA levels were assessed as described in Figure 4 A. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM ATRIP , BACH1 , and EXO1 splicing is upregulated in cells expressing wild-type BRCA1 but not S1423A-BRCA1, indicating that phosphorylation of BRCA1 S1423 is required for DNA damage-induced splicing. (E–G) FLAG and BCLAF1 ChIP-qRT-PCR analysis carried out in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt), or S1423A Flag-BRCA1 (S1423A). Cells were mock treated or treated with etoposide. Graphs represent the mean fold enrichment from three independent experiments ± SEM. (H) Representative western blots demonstrating wild-type and S1423A Flag-BRCA1 in HCC1937 cells used for splicing assays and ChIPs presented above. See also Figure S6 .

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Phospho-proteomics, Binding Assay, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: BRCA1/BCLAF1-Mediated mRNA Splicing Is Required for Maintenance of ATRIP, BACH1, and EXO1 Protein Expression (A) Representative western blots demonstrating that depletion of either BRCA1 or BCLAF1 results in downregulated expression of ATRIP, BACH1, and EXO1 proteins in response to DNA damage. (B) Representative western blot demonstrating DNA damage-dependent accumulation of ATRIP, BACH1, and EXO1 proteins over time following inhibition of proteosomal mediated protein degradation with MG132 (10 μM). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to MG132 treatment. (C) Representative western blots demonstrating DNA damage-dependent increased protein turnover in control and BRCA1- or BCLAF1-depleted cells following inhibition of protein translation with Cyclohexamide (10 μg/mL). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to Cyclohexamide treatment. (D) Representative western blots demonstrating BRCA1 and BCLAF depletion in cells used for experiments shown in (C). (E–G) Quantification of ATRIP, BACH1, and EXO1 protein levels shown in (C). Image densitometry values were normalized to 0 hr and decay curves fitted and used to calculate protein half-lives.

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Expressing, Western Blot, Inhibition, Control

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: BRCA1/BCLAF1-Mediated mRNA Splicing of ATRIP, BACH1, and EXO1 Promotes Resistance to DNA Damage and Efficient DNA Repair (A) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in BRCA1- or BCLAF1-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM. (B) Clonogenic survival assays demonstrating that depletion of BCLAF1 or U2AF65 induces sensitivity to IR in 293T cells. Mean surviving fraction of three independent experiments is plotted ± SEM. (C) Representative immunofluorescent staining of γ-H2AX marked DNA damage in untreated 293T cells depleted of either BCLAF1 or U2AF65 and 1 and 24 hr following 2Gy IR. (D. Quantification of three independent experiments described above (≥200 cells were scored/experiment). The mean fraction of cells containing ≥5 γ-H2AX foci is plotted ± SEM. Significant differences in the fraction of cells containing ≥5 γ-H2AX foci were assessed using Student’s two-tailed t test and are indicated by ∗∗∗ p < 0.001. (E) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in U2AF65-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM. See also Figure S7 .

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Expressing, Staining, Two Tailed Test

Journal: American Journal of Cancer Research

Article Title: Phospholipase C-ε regulates bladder cancer cells via ATM/EXO1

doi:

Figure Lengend Snippet: Gene expression profile in PLCε-silenced T24 cells and verification of selected DEGs. (A) RNA-seq mRNA expression data from Sanchez-Carbayo bladder dataset was used to compare PLCε expression between in BCa tumors (BCa) and normal bladder tissue (Bladder). (B) KEGG pathway enrichment analysis for the DEGs (> two-fold change and P < 0.05), the vertical axis was the pathway category, and the horizontal axis was the enrichment of pathways. (C) The significant GO category for indicated genes of mismatch repair between Ad-shPLCε and Ad-NC groups, the vertical axis was the GO terms, and the horizontal axis was the enrichment of GO, P value < 0.01 was used as a threshold to select significant GO categories. (D) Hierarchical clustering analysis of DEGs involved in DNA damage repair pathways. High expression levels are shown in green and low levels in red. (E, F) Detection gene levels of 4 selected DEGs by q-PCR in T24 and BIU-87 cells. The q-PCR data were normalized to the expression of β-actin (G) RNA-seq mRNA expression data from the TCGA to compare EXO1 expression between in BCa tumors (T) and their non-tumor counterparts (N). *P ≤ 0.05, **P ≤ 0.01, AND ***P < 0.001.

Article Snippet: The following primary antibodies were used: polyclonal rabbit antibody against PLCε (Santa Cruz, dilution 1:50), EXO1 (Elabscience, dilution 1:100), ATM (Abcam, 1:100).

Techniques: Gene Expression, RNA Sequencing, Expressing

Journal: American Journal of Cancer Research

Article Title: Phospholipase C-ε regulates bladder cancer cells via ATM/EXO1

doi:

Figure Lengend Snippet: High expression levels of PLCε and EXO1 in BCa tissues. (A, B) Disease free survival curves of BCa patients according to EXO1 and ATM mRNA levels in TCGA. (C) Representative haematoxyl in and eosin (H&E) staining and IHC staining in 72 BCa tissue samples and 24 corresponding adjacent non-neoplastic (BN) samples. Magnification × 200. Bars = 150 μm. H&E staining of bladder tissues were showed (a and i). Representative IHC staining of different staining intensities used as criteria in staining scoring: none staining was observed in BN (b-d), positive staining in BCa (j-l). PLCε localized in cytoplasm. EXO1 and ATM protein localized in nucleus. Images in the frame of (a-d and i-l) were enlarged to (e-h and m-p). (D-F) Average staining scores for PLCε, EXO1, and ATM expression in bladder tissues. (G, H) The correlation with PLCε and EXO1 expression and EXO1 and ATM expression in bladder tissues samples was analyzed by Pearson analysis. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001.

Article Snippet: The following primary antibodies were used: polyclonal rabbit antibody against PLCε (Santa Cruz, dilution 1:50), EXO1 (Elabscience, dilution 1:100), ATM (Abcam, 1:100).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: American Journal of Cancer Research

Article Title: Phospholipase C-ε regulates bladder cancer cells via ATM/EXO1

doi:

Figure Lengend Snippet: Correlation between PLCε, EXO1 and ATM expressions and the corresponding clinicopathological parameter in BCa patients

Article Snippet: The following primary antibodies were used: polyclonal rabbit antibody against PLCε (Santa Cruz, dilution 1:50), EXO1 (Elabscience, dilution 1:100), ATM (Abcam, 1:100).

Techniques:

Journal: American Journal of Cancer Research

Article Title: Phospholipase C-ε regulates bladder cancer cells via ATM/EXO1

doi:

Figure Lengend Snippet: A association between PLCε and EXO1 expression in BCa patients. A. PLCε and EXO1 protein expression in 24 BCa tissue samples (T) and 24 BN samples (N) were assayed by western blotting analysis. B, C. PLCε and EXO1 protein expression in tissues were quantified as mean optical density by Image J software. D, E. PLCε and EXO1 protein expression in tissues were assayed by Statistical analysis. F. Correlation curve of PLCε protein versus corresponding EXO1 by Statistical analysis. G. Western blotting analysis detected the expression of PLCε and EXO1 in two human BCa cell lines and SV-HUC-1 cells. H, I. Detection of PLCε and EXO1 by qPCR. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001.

Article Snippet: The following primary antibodies were used: polyclonal rabbit antibody against PLCε (Santa Cruz, dilution 1:50), EXO1 (Elabscience, dilution 1:100), ATM (Abcam, 1:100).

Techniques: Expressing, Western Blot, Software

Journal: American Journal of Cancer Research

Article Title: Phospholipase C-ε regulates bladder cancer cells via ATM/EXO1

doi:

Figure Lengend Snippet: PLCε depletion inhibit cell proliferation, induce G1 phase cell cycle arrest, apoptosis and cisplatin sensitivity. A. The T24 and BIU-87 cells were infected with adenovirus sh-PLCε, western blotting were detected the expression of PLCε. B. Western blotting analysis detected the expression of EXO1, cyclinD1, c-Myc, PCNA, BCL2, caspase 3, caspase 9 and BAX. C, D. Representative flow cytometry analysis of cell cycle phase distribution after 48 h of culture, compared to blank group. E-K. The T24 and BIU-87 cells were infected with vector-EXO1 for 72 h. E, F. Representative flow cytometry analysis of cell apoptosis after 72 h of culture in Ad-shPLCε or/and EXO1 groups. G, H. Clone formation analysis was employed to test the cell proliferation in Ad-shPLCε or/and EXO1 groups. I. IC50 values were measured by CCK-8 to compare the susceptibility of the three cell lines to cisplatin. J, K. IC50 values of T24 and BIU were measured by CCK-8 Analysis in Ad-shPLCε or/and EXO1 groups. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001.

Article Snippet: The following primary antibodies were used: polyclonal rabbit antibody against PLCε (Santa Cruz, dilution 1:50), EXO1 (Elabscience, dilution 1:100), ATM (Abcam, 1:100).

Techniques: Infection, Western Blot, Expressing, Flow Cytometry, Plasmid Preparation, CCK-8 Assay

Journal: American Journal of Cancer Research

Article Title: Phospholipase C-ε regulates bladder cancer cells via ATM/EXO1

doi:

Figure Lengend Snippet: PLCε regulates EXO1-mediated cell cycle, proliferation and apoptosis through nuclear translocation of p-ATM. A. The BCa cells were infected with vector-shEXO1, western blotting detected the expression of EXO1. B. Western blotting analysis detected the expression of EXO1, cyclinD1, c-Myc, PCNA, BCL2, caspase 3, caspase 9, and BAX in sh-NC and sh-EXO1 groups. C. The BCa cells were infected with vector-EXO1, western blotting detected the expression of EXO1. D. Western blotting analysis detected the expression of PLCε, EXO1, cyclinD1, c-Myc, PCNA, BCL2, caspase 3, caspase 9 and BAX in sh-EXO1 groups or/and Ad-shPLCε groups. E. Detection of p-ATM expression in nuclear and cytoplasm by western blot. F. Western blotting analysis showing that Ad-shPLCε reduced EXO1 level with 13 nM KU-55933 for 48 h. G. Immunofluorescence staining analysis demonstrating that Ad-shPLCε disrupted p-ATM nuclear translocation stimulated by cisplatin. Magnification: 400×. Bars = 100 μm. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001.

Article Snippet: The following primary antibodies were used: polyclonal rabbit antibody against PLCε (Santa Cruz, dilution 1:50), EXO1 (Elabscience, dilution 1:100), ATM (Abcam, 1:100).

Techniques: Translocation Assay, Infection, Plasmid Preparation, Western Blot, Expressing, Immunofluorescence, Staining

Journal: American Journal of Cancer Research

Article Title: Phospholipase C-ε regulates bladder cancer cells via ATM/EXO1

doi:

Figure Lengend Snippet: PLCε depletion enhanced the inhibitory effect of cisplatin on the growth of BCa cells. C-I. 8 μg/mL Cisplatin was treated in BCa cells for 48 h. A, B. Western blotting analysis showing the expressions of EXO1 and γ-H2AX stimulated by cisplatin at different concentrations (for 24 h) and different treat times (with 8 μg/mL) in BCa cells. C. Morphological change of bladder cancer cells with cisplatin or (and) Ad-shPLCε treatment. Magnification: 200×. Bars = 100 μm. D, E. Representative flow cytometry analysis of cell cycle phase distribution after 48 h of culture, compared to blank group. F. Western blotting detected the expression of cell cycle and proliferation related proteins. G, H. Clone formation analysis was employed to test the cell proliferation of BCa cell lines after treatment with cisplatin. I. Western blotting detected the expression of cell apoptosis related proteins. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001.

Article Snippet: The following primary antibodies were used: polyclonal rabbit antibody against PLCε (Santa Cruz, dilution 1:50), EXO1 (Elabscience, dilution 1:100), ATM (Abcam, 1:100).

Techniques: Western Blot, Flow Cytometry, Expressing

Journal: American Journal of Cancer Research

Article Title: Phospholipase C-ε regulates bladder cancer cells via ATM/EXO1

doi:

Figure Lengend Snippet: Decreased levels of serum miR-145 in BCa patients and PLCε was a direct target of miR-145 in BCa cells. A. Comparison of miR-145 levels in three cell lines by qPCR. vs SV-HUC-1. B. Expression levels of miR-145 in T24 and BIU cells transfected with miR-145 mimics and NC. C, D. PLCε mRNA and protein expression levels in the BCa cell lines detected by qRT-PCR and western blotting after transfection with miR-145 mimics. E. Schematic representation of the predicted miR-145 binding sites in 3’UTR of PLCε. F. Wild-type or mutant 3’UTR of PLCε gene reporter plasmid was co-transfected with miR-145mimics or NC into T24, followed by luciferase reporter assays. vs miR-NC. G. Changes of PLCε expression analysised by qPCR in BCa cells after treating with cisplatin or (and) miR-145. H, I. 8 μg/mL Cisplatin was treated in BCa cells for 48 h. H. Viability of BCa cells with cisplatin or (and) miR-145 mimics treatment detected by CCK-8. I. Western blotting analysis showing the expression of PLCε, EXO1, and γH2AX after cisplatin or (and) miR-145 mimics treatment for 48 h. J. The serum miR-145 expression in 40 patients with BCa, 19 patients with urinary benign diseases (BBD) and 21 healthy controls (Normal) were obtained by qPCR. Expression levels of miR-145 were normalized to U6. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001.

Article Snippet: The following primary antibodies were used: polyclonal rabbit antibody against PLCε (Santa Cruz, dilution 1:50), EXO1 (Elabscience, dilution 1:100), ATM (Abcam, 1:100).

Techniques: Comparison, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Binding Assay, Mutagenesis, Plasmid Preparation, Luciferase, CCK-8 Assay

Journal: American Journal of Cancer Research

Article Title: Phospholipase C-ε regulates bladder cancer cells via ATM/EXO1

doi:

Figure Lengend Snippet: A mechanism for PLCε depletion-mediated cisplatin sensitivity of BCa. Schematic diagram describing the functional significance of PLCε in BCa cells. PLCε regulate EXO1 medicated-cell cycle, proliferation, apoptosis and cisplatin sensitivity through nuclear translocation of p-ATM and can be regulated by miR-145.

Article Snippet: The following primary antibodies were used: polyclonal rabbit antibody against PLCε (Santa Cruz, dilution 1:50), EXO1 (Elabscience, dilution 1:100), ATM (Abcam, 1:100).

Techniques: Functional Assay, Translocation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: 5‐Azacytydine and resveratrol reverse senescence and ageing of adipose stem cells via modulation of mitochondrial dynamics and autophagy

doi: 10.1111/jcmm.13914

Figure Lengend Snippet: Sequences of primers used in qPCR

Article Snippet: Next, the membrane was washed with TBST and incubated overnight with primary antibody for: mitofusin 1 (MFN) (orb11040; Biorbyt, Cambridge, UK), PINK (orb331233; Biorbyt), caspase‐3 (437800; Life Technologies), beta‐actin (A5441; Sigma‐Aldrich) and mitochondrial fision factor ‐MFF (orb325479; Biorbyt) at a dilution of 1:500.

Techniques: Sequencing, Amplification

Journal: Journal of Cellular and Molecular Medicine

Article Title: 5‐Azacytydine and resveratrol reverse senescence and ageing of adipose stem cells via modulation of mitochondrial dynamics and autophagy

doi: 10.1111/jcmm.13914

Figure Lengend Snippet: AZA / RES treatment improved mitochondrial condition and reduced ROS accumulation in ASC EMS . To investigate oxidative stress in cells, they were culture for 24 hours in control or experimental conditions and subjected to further analysis. MMP was measured using JC ‐1 probe by flow cytometry (A). MMP depolarization in ASC EMS was markedly reversed in ASC EMS II (B). Intracellular ROS level was stained with H2 DCFDA and detected by flow cytometry. Results were respectively shown in the histograms (A). AZA / RES treatment significantly reduced ROS production in ASC EMS (C). Antioxidative properties of cells were analyzed by spectrophotometric measurement of SOD activity (D). ASC EMS II displayed enhanced enzyme activity. Using RT ‐ PCR amount of miR‐24 was evaluated (E). TEM analysis showed ultrastructural changes in mitochondria morphology in ASC EMS and it reversal after AZA / RES treatment (F). Boxed regions were magnified and showed on the left side. Based on representative photographs, quantification of aberrant mitochondria was performed (G). Mitochondrial fission was examined using RT ‐ PCR for FIS (H) while mitophagy was established by measuring the expression of PINK (I) and PARKIN (J). Moreover, we established that AZA / RES diminished expression of Mief1 (K) and Mief2 (L). Furthermore, using western blot amount of MFF , MNF , PINK were investigated (M). In treated cells, AZA / RES mitigated both, mitochondrial division and mitophagy. Scale bars: 1 μm. Results expressed as mean ± SD . Statistical significance indicated as asterisk (*) when comparing the result to ASC CTRL , and as hashtag (#) when comparing to ASC EMS . # P < 0.05; ##, ** P < 0.01; ###, *** P < 0.001

Article Snippet: Next, the membrane was washed with TBST and incubated overnight with primary antibody for: mitofusin 1 (MFN) (orb11040; Biorbyt, Cambridge, UK), PINK (orb331233; Biorbyt), caspase‐3 (437800; Life Technologies), beta‐actin (A5441; Sigma‐Aldrich) and mitochondrial fision factor ‐MFF (orb325479; Biorbyt) at a dilution of 1:500.

Techniques: Control, Flow Cytometry, Staining, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: 5‐Azacytydine and resveratrol reverse senescence and ageing of adipose stem cells via modulation of mitochondrial dynamics and autophagy

doi: 10.1111/jcmm.13914

Figure Lengend Snippet: AZA / RES diminish apoptosis and autophagy in ASC EMS by the inhibition of mitochondrial fission. In order to uncover the mechanism underlying AZA / RES beneficial effects on cells, they were cultured in control, AZA / RES and/or mitochondrial division inhibitor‐ MDIVI ‐, supplemented medium. After 24 hours of propagation cells were subjected to RT ‐ PCR , TEM and confocal analysis. Using RT ‐ PCR expression of p53 (A), p21 (B), p62 (C), PINK (D) and MFN (E) was analyzed. Mitochondrial dynamics and morphology of mitochondrial net were further visualized using TEM and confocal microscope (MitoRed staining) (F). Boxed regions were enlarged and presented below MitoRed staining to visualize shape of mitochondrial network. Obtained results indicated that beneficial effects of AZA / RES result from inhibition of mitochondrial fission and modulation of autophagy in treated cells. Scale bars: TEM : 5 μm, confocal: 20 μm. Results expressed as mean ± SD . Statistical significance indicated as asterisk (*) when comparing the result to ASC CTRL , and as hashtag (#) when comparing to ASC EMS . #, * P < 0.05; ##, ** P < 0.01; ###, *** P < 0.001

Article Snippet: Next, the membrane was washed with TBST and incubated overnight with primary antibody for: mitofusin 1 (MFN) (orb11040; Biorbyt, Cambridge, UK), PINK (orb331233; Biorbyt), caspase‐3 (437800; Life Technologies), beta‐actin (A5441; Sigma‐Aldrich) and mitochondrial fision factor ‐MFF (orb325479; Biorbyt) at a dilution of 1:500.

Techniques: Inhibition, Cell Culture, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Microscopy, Staining