Journal: Cardiovascular Research

Article Title: LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

doi: 10.1093/cvr/cvu114

Figure Lengend Snippet: LOX-1 inhibition reduces ROS generation, autophagy, mtDNA damage, and NLRP3 inflammasome expression. (A) LOX-1 inhibition by pre-treatment with a specific antibody or siRNA transfection blocks LOX-1 expression. (B) LOX-1 inhibition reduces cellular ROS generation. (C) LOX-1 inhibition decreases autophagic flux. (D and E) LOX-1 inhibition protects mtDNA from damage and inhibits NLRP3 expression. Cells were pre-treated with 10 μg/mL of LOX-1 antibody (Ab) or transfected with 20 nM of siRNA directed at LOX-1 for 24 h; then the cells were treated with 10 ng/mL of LPS for another 24 h. ssc, scrambled siRNA control. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Article Snippet: Briefly, cells were treated with the siRNA duplex solution for 24 h. The medium was then replaced with normal culture medium, and cells were treated with 10 ng/mL of LPS for another 24 h. As control, cells were transfected with sequence scrambled siRNA control (ssc, Santa Cruz).

Techniques: Inhibition, Expressing, Transfection, Control

Journal: Cardiovascular Research

Article Title: LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

doi: 10.1093/cvr/cvu114

Figure Lengend Snippet: DNase II knockdown enhances the expression of LOX-1 and NLRP3 inflammasome, and aggravates mtDNA damage. (A) Expression of DNase II is inhibited by siRNA transfection. (C–E) DNase II knockdown enhances ROS generation, mtDNA damage, autophagy, and expression of NLRP3. Cells were transfected with 20 nM DNase II siRNA for 24 h, then the cells were treated with 10 ng/mL of LPS for another 24 h. ssc, scrambled siRNA control. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Article Snippet: Briefly, cells were treated with the siRNA duplex solution for 24 h. The medium was then replaced with normal culture medium, and cells were treated with 10 ng/mL of LPS for another 24 h. As control, cells were transfected with sequence scrambled siRNA control (ssc, Santa Cruz).

Techniques: Knockdown, Expressing, Transfection, Control

Journal: Cardiovascular Research

Article Title: LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

doi: 10.1093/cvr/cvu114

Figure Lengend Snippet: Study in mice primary peritoneal macrophages confirms the results in THP-1 cells. (A and B) LOX-1 knockdown inhibits mtDNA damage and expression LC3-II, P62, and NLRP3 inflammasome expression. Cells were transfected with LOX-1 siRNA for 24 h, then the cells were treated with LPS for another 24 h. (C to E) Both ROS inhibitors apocynin and YCG063 decrease mtROS generation, mtDNA damage, as well as expression of LC3-II, P62, LOX-1, and NLRP3 inflammasome expression. Cells were treated with LPS for 24 h in the presence or absence of 0.01 mM apocynin and 10 µM YCG063. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05. (F) Proposed signalling pathway linking LOX-1 to mtDNA damage, autophagy, and NLRP3 activation. There appears to be a positive feedback loop between LOX-1 and ROS. Activation of both LOX-1 and ROS induces mtDNA damage. Though most of damaged DNA can be removed by autophagy and DNase II degradation, some damaged mtDNA that persists may result in activation of NLRP3 inflammasome.

Article Snippet: Briefly, cells were treated with the siRNA duplex solution for 24 h. The medium was then replaced with normal culture medium, and cells were treated with 10 ng/mL of LPS for another 24 h. As control, cells were transfected with sequence scrambled siRNA control (ssc, Santa Cruz).

Techniques: Knockdown, Expressing, Transfection, Activation Assay

Journal: Cardiovascular Research

Article Title: LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

doi: 10.1093/cvr/cvu114

Figure Lengend Snippet: LPS and mtROS generation. (A) LPS induces mtROS generation in a dose-dependent manner. Cells were treated with 1–100 ng/mL of LPS for 24 h before measurement of mtROS. (B) LOX-1 knockdown by antibody or siRNA transfection inhibits mtROS generation. Cells were pre-treated with 10 μg/mL of LOX-1 antibody (Ab) or transfected with 20 nM its siRNA for 24 h, then the cells were treated with 10 ng/mL of LPS for another 24 h. (C) ROS inhibitors apocynin and YCG063 inhibit mtROS generation. Cells were treated with 10 ng/mL of LPS for 24 h in the presence or absence of 1 mM apocynin and 10 µM YCG063. (D) Autophagy inducer rapamycin inhibits, while autophagy inhibitor 3-methyladenine enhances mtROS generation. Cells were treated with 10 ng/mL of LPS for 24 h in the presence or absence of 10 nM rapamycin and 5 mM 3-methyladenine. (E) DNase II knockdown by its siRNA transfection increases mtROS generation. Cells were transfected with 20 nM DNase II siRNA for 24 h, then the cells were treated with 10 ng/mL of LPS for another 24 h. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Article Snippet: Briefly, cells were treated with the siRNA duplex solution for 24 h. The medium was then replaced with normal culture medium, and cells were treated with 10 ng/mL of LPS for another 24 h. As control, cells were transfected with sequence scrambled siRNA control (ssc, Santa Cruz).

Techniques: Knockdown, Transfection

Journal: Advanced Science

Article Title: A microRNA Cluster Controls Fat Cell Differentiation and Adipose Tissue Expansion By Regulating SNCG

doi: 10.1002/advs.202104759

Figure Lengend Snippet: Global transcriptomics reveal that the miR‐424(322)/503 cluster regulates multiple genetic programs related to adipocyte function. RNA sequencing (RNA‐seq) was performed on engineered human adipocytes with increased levels of the cluster miR‐424(322)/503 A) over the course of differentiation (14 days) and B) during the last 48 h of terminal differentiation. Upper left: principal component analysis (PCA). Upper right and down: gene set enrichment analysis (GSEA) was used to uncover enriched gene signatures from the molecular signatures database (MSigDB). Red and blue circles (nodes) represent different annotations exhibiting positive or negative enrichment, respectively (FDR q ‐val < 0.05). Size of nodes reflect p ‐value ( p ‐val < 0.05 for all samples). Arrows interconnect pathways presenting common genes in the leading edge subset for each annotation. C) Venn diagram showing the overlap between potential Targetscan (TS) target genes with differential expressions between Wt/miR‐KO mouse adipocytes (red circle); differentiating human adipocytes with or without induction of the miR‐424(322)/503 cluster (green circle); and adipocytes with or without cluster induction for the last 48 h of differentiation (blue circle). D) Heatmaps show hierarchical clustering analysis for the 24‐gene signature predicted to be regulated by the miR‐424(322)/503. Expression of target gene candidates on E) the 0, 7th and 14th day of the course of adipocyte differentiation ( n = 4/time‐point; One‐way ANOVA), and in F) adipose‐derived mesenchymal stem cells (ASC52telo)‐derived adipocytes challenged with an antagomiR directed against the miR‐424 ( n = 6/group; Student's t ‐test). Data are presented as mean ± S.E.M. PKDCC did not show qRT‐PCR amplification data. * p < 0.05; ** p < 0.01; *** p < 0.001. NS, not significant.

Article Snippet: For the main purpose of additional testing and validation of previous results in primary cells, human telomerase reverse transcriptase‐immortalized adipose‐derived mesenchymal ASC52telo stem cells (SCRC‐4000, ATCC, LGC Standards SLU, Barcelona, Spain) [ ] were cultured in a Mesenchymal Stem Cell Basal Medium (ATCC, PCS‐500‐030) plus FBS (2%), recombinant human (rh)FGF basic (5 ng mL −1 ), rhFGF acidic (5 ng mL −1 ), rhEGF (5 ng mL −1 ), L‐Alanyl‐L‐Glutamine (2.4 m m ) and G418 (0.2 mg mL −1 ) at 37 °C in a 5% CO2 in air atmosphere.

Techniques: RNA Sequencing, Expressing, Derivative Assay, Quantitative RT-PCR, Amplification

Journal: Cancer discovery

Article Title: TOX regulates growth, DNA repair, and genomic instability in T-cell Acute Lymphoblastic Leukemia

doi: 10.1158/2159-8290.CD-17-0267

Figure Lengend Snippet: (a) Flow cytometry analysis showing DNA content of zebrafish T-ALL (blue) compared to normal blood-derived DNA (red). Representative leukemias are shown with changes in DNA index noted (DI). 2n has a DNA index of 1. (b) Quantization of DNA content. Asterisk denotes p=0.013, Fisher Exact Test. (c) Genomic DNA alterations identified by whole genome sequencing. The log-log scatter plot represents read counts within non-overlapping window size of 10 kb across the genome comparing leukemia and control tissue from the same, representative animal. Amplifications found in the MYC+TOX leukemia are denoted by the green circle. (d) Manhattan plot representing the copy number variation across the genome of a representative MYC (top) and MYC+TOX (bottom) expressing T-ALL. Regions of significant gain, amplification (AMPL), high level amplification (HLAMPL), and neutral 2N copy number (NEUT) noted. (e-f) Analysis of metaphase spreads from MEF cells infected with control (MOCK), full-length Wild-type TOX (TOX WT), or TOX that lacks the HMG box domain (TOX dHMG). Arrows denote chromosome abnormalities. (f) Quantification of cells with genomic abnormalities. >50 nuclei were counted per condition and replicated three independent times. Error bars denote standard deviation. *, p<0.05 and **, p<0.005, Student’s t-test. Not significant (NS).

Article Snippet: NIH3T3 and MEF cells were obtained from ATCC in 2012 and 2015, respectively and used within three months of receipt.

Techniques: Flow Cytometry, Derivative Assay, Sequencing, Control, Expressing, Amplification, Infection, Standard Deviation

Journal: bioRxiv

Article Title: Developmental-stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors

doi: 10.1101/356527

Figure Lengend Snippet: A. Explanted retinae were cultured on membrane, transduced with lentiviral sh RB1 or sh SCR control shRNA vectors, and EdU-labeled for 4 h immediately prior to harvest. Shading in the intermediate and central retina depicts progressively higher maturation of ARR3+ cone precursors. Dashed line represents a section across the cultured retina that may be used to compare cone precursor behaviors according to topographic position. B. Lentiviral constructs used for intact retina transductions. C-E. Maturation-related cell cycle entry at 12 days post-RB KD in week 18 human retina examined in peripheral or central retina (as in panel 1A) or in a transition zone displaying the most peripheral ARR3+ cells. Each image is divided into four panels to assess cone markers (RXRβ or ARR3, white), cell cycle markers (Ki67 or EdU, red), lentivirus transduced cells (YFP, green) and DAPI-stained nuclei (blue). Scale bars, 20 μm. F. Percentages of Ki67+ or EdU+ cells (stained separately in adjacent sections) among RXRγ+,YFP+ cone precursors in peripheral, intermediate, and central retina. Error bars, standard deviation (SD). Significance assessed by t-test (*, p<0.01, **, p<0.001).

Article Snippet: We used pLKO.1C-YFP- shRBL -733 ( ) for human RB KD and pLKO.1C-YFP-sh RB1 (with targeting sequence 5’-AACGGACGTGTGAACTTATAT-3’ ( )) for mouse Rb1 KD. pLKO.1C-scrambled (sh SCR ) expressing a non-targeting control shRNA was Addgene plasmid 1864.

Techniques: Cell Culture, Membrane, Transduction, Control, shRNA, Labeling, Construct, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Developmental-stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors

doi: 10.1101/356527

Figure Lengend Snippet: sh SCR transduced retina at 12 DIC shows Ki67+,RXRγ(-) cells in the neuroblasti’c layer, similar to that observed in peripheral shRbl -transduced retina . Scale bar, 20μm.

Article Snippet: We used pLKO.1C-YFP- shRBL -733 ( ) for human RB KD and pLKO.1C-YFP-sh RB1 (with targeting sequence 5’-AACGGACGTGTGAACTTATAT-3’ ( )) for mouse Rb1 KD. pLKO.1C-scrambled (sh SCR ) expressing a non-targeting control shRNA was Addgene plasmid 1864.

Techniques:

Journal: bioRxiv

Article Title: Developmental-stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors

doi: 10.1101/356527

Figure Lengend Snippet: A. Cone-specific Rb1 knockout in RGP-Cre;Rb1 lox/lox but not in littermate Rb1 lox/lox mouse retinae at postnatal day (P) 10. Upper panels show sections traversing the outer nuclear layer (ONL) and inner nuclear layer (INL). Boxed ONL regions are enlarged in lower panels. White arrows show Rxry+ cones and yellow arrows show Müller glia. B. Number of Arr3+ or L/M-Opsin+ cells analyzed for Ki67 expression at various ages in RGP-Cre,Rb1 lox/lox retinae. C. P9 explanted retinae transduced with pLKO-sh Rb1 or pLKO- SCR control lentivirus stained for Rb, Rxry and YFP at 14 DIC. Arrows show Rb staining in sh SCR -transduced but not in sh Rb1 -transduced YFP+,Rxry+ cone precursors. D. Number of Arr3+,YFP+ cells analyzed for Ki67 expression and EdU incorporation at 7 and 14 days post Rb KD. E. P4-explanted and sh RB1 -transduced mouse retina co-immunostained for cone markers Rxry or Arr3 (white), sh Rb1 -transduction marker YFP (green), and proliferation markers Ki67 or EdU (red). Examples of ONL Rxrγ+,YFP+,Ki67+ (i), Rxrβ+,YFP+,EdU+ (ii), and Arr3+,YFP+,Ki67- (iii) cells at 7 and 14 DIC are shown. White arrows, proliferation-marker-positive cone precursors; yellow arrows, proliferation-marker-negative cone precursors; blue arrows, proliferation-marker-positive Arr3-negative cells. F. Quantitation of YFP+,Rxrβ+ or YFP+,Arr3+ cells co-stained for Ki67 or EdU from explanted P4 retinae transduced with sh Rb1 or sh SCR and analyzed at 7 DIC, 14 DIC, and 21 DIC. Error bars, SD. Significance was assessed by ANOVA with Tukey HSD Post-hoc Test. (*, p<0.05,**, p<0.001). Scale bars in A, C, E, 10 μm.

Article Snippet: We used pLKO.1C-YFP- shRBL -733 ( ) for human RB KD and pLKO.1C-YFP-sh RB1 (with targeting sequence 5’-AACGGACGTGTGAACTTATAT-3’ ( )) for mouse Rb1 KD. pLKO.1C-scrambled (sh SCR ) expressing a non-targeting control shRNA was Addgene plasmid 1864.

Techniques: Knock-Out, Expressing, Transduction, Control, Staining, Marker, Quantitation Assay

Journal: bioRxiv

Article Title: Developmental-stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors

doi: 10.1101/356527

Figure Lengend Snippet: A. Developmental increase in MYCN expression in human but not in mouse cone precursors. Upper panels , ARR3+ cells in human week 15 retinae show increasing MYCN expression from the mid-periphery to parafovea. Lower panels , Mycn was weakly expressed in Arr3+ cells at P10 and not detected above background levels at P20 in both WT and RGP-MDM2;RGP-Cre;Rb lox/lox mice retinae demonstrating that the MDM2 transgene did not alter endogenous Mycn expression. B. Quantitation of MDM2 expression in Rxrγ+ cone precursors in cultured vs. in vivo developed RGP-MDM2 littermate retinae. MDM2 levels were compared between P6 retina and P4 retinal explants cultured for 2 days or between P9 retinae and P4 explants cultured for 5 days. Each dot represents a quantitatively imaged cell outlined by Rxrγ signal, and box plots show median (line inside box), upper and lower quartiles (box borders), and data range (whiskers). Significance was assessed by t-test. P values are as shown; ns, not significant. C. Rb1 KD in a P4+7 DIC retinal explant co-stained for Rxrg, Rb, and YFP. White arrows, infected (YFP+) cells that are Rb+ after transduction with sh SCR but not with sh Rb1 . Yellow arrow, uninfected (YFP-) cell with Rb expression. D. Lentiviral constructs for transduction of intact murine retina with ectopic Mycn or Mycn T58A (BE-Neo-Mycn (T58A) ) or with the empty vector (BE-Neo). E. Mycn expression in Rxrγ+ cells of P4 retinal explants transduced with Mycn T58A at 7 DIC. F. Quantitation of Mycn levels in Rxrγ+ cells ( left ) or Arr3+ ( right ) at 7 DIC in P4 and P9 explants, respectively. Box plots and significance tests are as in panel B. Scale bars in A, C, and E, 10 μm.

Article Snippet: We used pLKO.1C-YFP- shRBL -733 ( ) for human RB KD and pLKO.1C-YFP-sh RB1 (with targeting sequence 5’-AACGGACGTGTGAACTTATAT-3’ ( )) for mouse Rb1 KD. pLKO.1C-scrambled (sh SCR ) expressing a non-targeting control shRNA was Addgene plasmid 1864.

Techniques: Expressing, Quantitation Assay, Cell Culture, In Vivo, Staining, Infection, Transduction, Construct, Plasmid Preparation

Journal: bioRxiv

Article Title: Developmental-stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors

doi: 10.1101/356527

Figure Lengend Snippet: A. Experimental design for lentiviral transductions of WT or RGP-MDM2 retinae. All retinae were co-transduced with sh Rb1 or sh SCR , and with BE-Neo- Mycn T58A or BE-Neo vector. B. Number of YFP+, Arr3+ cells from P9 RGP-MDM2 explanted retinae analyzed for Ki67 at 7 and 14 days post co-transduction with sh Rb1 and BE-Neo-Mycn T58A C, D. Quantitation of YFP+,Rxry+ cells co-stained for Ki67 or EdU in P4 WT or RGP-MDM2 transgenic retinal explants cultured for 7 or 14 days post-transduction. Labels indicate oncogenic variables (sh Rb1 , MDM2, Mycn) and omit the applied control vectors or WT genotype. E. Quantitation of YFP+,Rxry+ cells or YFP+,Arr3+ cells co-stained with Ki67 or EdU from P4 RGP-MDM2 retinal explants transduced with sh Rb1 and BE-Neo-Mycn T58A and analyzed at 7 DIC, 14 DIC, and 21 DIC. Error bars, SD. Significance assessed by ANOVA with Tukey HSD Post-hoc Test (*, p<0.05); **, p<0.001, ns=not significant).

Article Snippet: We used pLKO.1C-YFP- shRBL -733 ( ) for human RB KD and pLKO.1C-YFP-sh RB1 (with targeting sequence 5’-AACGGACGTGTGAACTTATAT-3’ ( )) for mouse Rb1 KD. pLKO.1C-scrambled (sh SCR ) expressing a non-targeting control shRNA was Addgene plasmid 1864.

Techniques: Transduction, Plasmid Preparation, Quantitation Assay, Staining, Transgenic Assay, Cell Culture, Control

Journal: bioRxiv

Article Title: Developmental-stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors

doi: 10.1101/356527

Figure Lengend Snippet: A-D. Ki67 expression (A, C) and EdU incorporation (B, D) (red) at 30 DIC (A, B) and 74 DIC (C, D) after transduction of week 18 fetal retina. Dashed circles identify ring-like arrangement of RXRγ+ nuclei resembling rosettes. Arrows show proliferating non-cone (RXRγ-) cells, which were either transduced (yellow arrows) or non-transduced (white arrows) and likely represent progenitors or glia. E. Quantitation of Ki67 expression and EdU incorporation in RXRγ+ cone precursors in explanted week 18 retinae from 12 to 123 DIC. Data at each age is from central, intermediate and peripheral regions. Error bars, SD. Significance assessed by ANOVA with Tukey HSD Post-hoc Test (*, p<0.05). F-J , Hematoxylin and eosin (H&E) staining of human week 18 retina at the indicated DIC after transduction with sh RB1 (F, G, H, J) or sh SCR (I). Dashed circles identify Flexner-Wintersteiner rosettes (F-H) or fleurettes (J, boxed regions at left enlarged at right) . K . Predominance of RXRγ+,YFP+ cells in the same retinoma-like region as shown in panel J. Dashed yellow line separates proliferating ( upper left ) and nonproliferating ( lower right ) regions. L. H&E staining ( upper left ) performed after p16 INK4A immunostaining and imaging of RB-depleted retina at 123 DIC, demonstrating central fleurette with predominantly cytoplasmic p16 in RXRγ lo cells (white arrows) and nuclear p16 in RXRγ hi cells (yellow arrows). M. Another representative fleurette at 123 DIC, composed of RXRγ+,Ki67− cells. Scale bars, 20 μm.

Article Snippet: We used pLKO.1C-YFP- shRBL -733 ( ) for human RB KD and pLKO.1C-YFP-sh RB1 (with targeting sequence 5’-AACGGACGTGTGAACTTATAT-3’ ( )) for mouse Rb1 KD. pLKO.1C-scrambled (sh SCR ) expressing a non-targeting control shRNA was Addgene plasmid 1864.

Techniques: Expressing, Transduction, Quantitation Assay, Staining, Immunostaining, Imaging

Journal: bioRxiv

Article Title: Developmental-stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors

doi: 10.1101/356527

Figure Lengend Snippet: A,B,E,F. Quantitation of p16 and p130 protein in RXRγ+,YFP+ cells in human retina at 6, 12, 30, and 74 days post-transduction with sh RB1 or sh SCR (A,B), and in P4 WT or RGP-MDM2 mouse retinae at 7, 14, and 21 days posttransduction with sh SCR , sh Rb1 , BN vector, BN- Mycn T58A (E,F), based on immunostaining shown in panels (C,D and G,H). Each dot represents signal in a quantitatively imaged RXRγ+,YFP+ cell outlined by RXRγ signal. Box plots show median (line inside box), upper and lower quartiles (box borders), and data range (whiskers). Significance assessed by t-test (**,## p>0.0001; ns=not significant). C,D,G,H. Immunofluorescence staining of p16 (C, G) and p130 (D, H) in human retina at 6, 12, and 30 days post-transduction with sh RB1 or sh SCR (C,D) and in P4 WT or RGP-MDM2 mouse retinae at 7 and 14 days post-transduction with (G,H). Scale bars, 20 μm.

Article Snippet: We used pLKO.1C-YFP- shRBL -733 ( ) for human RB KD and pLKO.1C-YFP-sh RB1 (with targeting sequence 5’-AACGGACGTGTGAACTTATAT-3’ ( )) for mouse Rb1 KD. pLKO.1C-scrambled (sh SCR ) expressing a non-targeting control shRNA was Addgene plasmid 1864.

Techniques: Quantitation Assay, Transduction, Plasmid Preparation, Immunostaining, Immunofluorescence, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Four-octyl itaconate activates Keap1-Nrf2 signaling to protect neuronal cells from hydrogen peroxide

doi: 10.1186/s12964-018-0294-2

Figure Lengend Snippet: Nrf2 activation mediates 4-octyl itaconate-induced neuronal cell protection against H 2 O 2 . SH-SY5Y cells ( a - e ) or the primary murine neurons ( i - k ), with the applied Nrf2 shRNA or the scramble control shRNA (“shC”), were either untreated or treated with 4-octyl itaconate (OI), mRNA and protein expression of listed genes were shown ( a - c , and i ); Cells were pretreated for 30 min with OI (25 μM), followed by stimulation of H 2 O 2 (300 μM) for indicated time, cell viability (CCK-8 OD, d ), cell death (LDH release, j ) and apoptosis (TUNEL ratio increase, e , and k ) were tested. Stable SH-SY5Y cells, with the CRISPR/Cas9-Nrf2 KO construct (“Nrf2-KO”) or the CRISPR/Cas9 control construct (“Cas9-c”), were treated with 4-octyl itaconate (OI), listed proteins were shown ( f ); Cells were pretreated for 30 min with OI (25 μM), followed by stimulation of H 2 O 2 (300 μM) for indicated time, cell viability ( g ) and apoptosis ( h ) were tested. Expression of listed proteins were quantified and normalized to the loading control ( c , f and i ). “shNrf2 (m)” stands for murine Nrf2 shRNA ( I - K ). Bars stand for mean ± standard deviation (S.D., n = 5). # P < 0.05 vs. “shC” cells ( a , b , d and e ). # P < 0.05 ( g , h , j and k )

Article Snippet: Two different lentivirus-packed Nrf2 shRNAs, targeting non-overlapping sequence of human Nrf2 (sc-37030-V/“shNrf2–1” and sc-44332-V/“shNrf2–2”) , as well as the lentiviral murine Nrf2 shRNA [sc-37049-V, “shNrf2 (m)”] and the scramble nonsense control shRNA (“shC”, sc-108080) were purchased from Santa Cruz Biotech (Santa Cruz, CA). shRNA lentivirus were added to cultured cells in the presence of polybrene (5 μg/mL) for 48 h. Puromycin (1.0 μg/mL) was then included to select stable cells for 4–5 passages.

Techniques: Activation Assay, shRNA, Control, Expressing, CCK-8 Assay, TUNEL Assay, CRISPR, Construct, Standard Deviation

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

doi: 10.1002/ijc.29114

Figure Lengend Snippet: Western Blotting and chromosome aberration analysis. ( a ) Western Blotting analysis for protein expression; ( b ) Frequencies of chromatid breaks 48 hr after transfection with siRNA against Bmi-1 and scramble RNA (siControl). Two hundred metaphases were analyzed. Error bars indicate standard deviation (SD) and ( c ) Examples of chromatid breaks (indicated by arrows) in HeLa cells.

Article Snippet: Small interfering RNAs (siRNA) against human Bmi-1, cylin E or control siRNA (scramble RNA sequence) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Expressing, Transfection, Standard Deviation

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

doi: 10.1002/ijc.29114

Figure Lengend Snippet: Bmi-1 knockdown-induced chromatid breaks were rescued by knockdown of cyclin E. ( a ) Western Blotting analysis confirmed the effective knockdown of cylin E and ( b ) Frequencies of chromatid breaks 48 hr after transfection with siRNA against Bmi-1 together with siRNA against cyclin E or scramble RNA. Two hundred metaphases were analyzed. Error bars indicate SD.

Article Snippet: Small interfering RNAs (siRNA) against human Bmi-1, cylin E or control siRNA (scramble RNA sequence) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, Western Blot, Transfection

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

doi: 10.1002/ijc.29114

Figure Lengend Snippet: Bmi-1 knockdown-induced chromatid breaks were rescued by ectopic p21 overexpression. ( a ) Western Blotting analysis confirmed the effective p21 overexpression and ( b ) Frequencies of chromatid breaks 48 hr after transfection with siRNA against Bmi-1 together with pCEP-p21 or empty vector. Two hundred metaphases were analyzed. Error bars indicate SD.

Article Snippet: Small interfering RNAs (siRNA) against human Bmi-1, cylin E or control siRNA (scramble RNA sequence) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, Over Expression, Western Blot, Transfection, Plasmid Preparation

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

doi: 10.1002/ijc.29114

Figure Lengend Snippet: Bmi-1 knockdown increased the proportions of cells with DNA synthesis and replicative stress. ( a ) Examples of BrdU-staining to indicate the cells with DNA synthesis; ( b ) Frequencies of BrdU-positive cells after transfection with siRNA against Bmi-1 or scramble RNA; 500 cells were analyzed and error bars indicate SD; (c) Example of pan-nuclear staining of γ-H2AX (indicated by arrows) and ( d ) Frequencies of pan-nuclear γ-H2AX-positive cells after transfection with scramble siRNA, siRNA against Bmi-1, the combination of siRNA against Bmi-1 and empty vectors, or the combination of siRNA against Bmi-1 and p21 overexpression vectors. Five hundred cells were analyzed and error bars indicate SD. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]

Article Snippet: Small interfering RNAs (siRNA) against human Bmi-1, cylin E or control siRNA (scramble RNA sequence) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, DNA Synthesis, BrdU Staining, Transfection, Staining, Over Expression