stat3  (Bio-Techne corporation)

Journal: PLOS ONE

Article Title: Investigation of the anti-tumor mechanism of tirabrutinib, a highly selective Bruton’s tyrosine kinase inhibitor, by phosphoproteomics and transcriptomics

doi: 10.1371/journal.pone.0282166

Figure Lengend Snippet: Antiproliferative activity (A) and BTK autophosphorylation inhibitory effects (B) of tirabrutinib. (A) TMD8 or U-2932 cells were treated with vehicle or different concentrations of tirabrutinib and incubated for 72 h at 37°C, 5% CO 2 /95% air. After culturing, the growth inhibition rate (%) was calculated by measuring a luminescent signal proportional to the amount of intracellular ATP using the CellTiter-Glo Luminescent Cell Viability Assay. The growth inhibition rate in the tirabrutinib group is plotted as the mean of 3 or 4 treated cultures from each treatment group ± standard error for TMD8 or U-2932, respectively. (B) TMD8 or U-2932 cells were treated with vehicle or different concentrations of tirabrutinib and incubated for 4 h at 37°C, 5% CO 2 /95% air. Autophosphorylated BTK (p-BTK) and total BTK (BTK) proteins were detected by western blot analysis. The intensity of each western blot band shown in was determined. The ratio of phosphorylated to total BTK is described as the mean of three cases ± standard error in the bar chart. The symbol -∞ indicates the vehicle group. The curve was estimated by nonlinear regression analysis using a four-parameter logistic model. BTK, Bruton’s tyrosine kinase.

Article Snippet: Rabbit monoclonal anti- phosphorylated BTK (Tyr-223) antibody (p-BTK; Novus Biologicals, #NB100-79907), rabbit monoclonal BTK antibody (Cell Signaling Technology, Inc., #3533), rabbit polyclonal phosphorylated AKT (Ser-473) antibody (p-AKT; Cell Signaling Technology, Inc., #9271), rabbit monoclonal AKT (pan) (C67E7) antibody (Cell Signaling Technology, Inc., #4691), rabbit polyclonal phosphorylated p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204) antibody (p-ERK1/2; Cell Signaling Technology, Inc., #9101), rabbit monoclonal p44/42 MAPK (ERK1/2) (137F5) antibody (Cell Signaling Technology, Inc., #4695), rabbit monoclonal phosphorylated PLCγ2 (Tyr-759) (E9E9Y) antibody (p-PLCγ2; Cell Signaling Technology, Inc., #50535), rabbit monoclonal PLCγ2 (E5U4T) antibody (Cell Signaling Technology, Inc., #55512), mouse monoclonal phosphorylated IκBα (Ser-32/36) (5A5) antibody (p-IκBα; Cell Signaling Technology, Inc., #9246), rabbit monoclonal IκBα (44D4) antibody (Cell Signaling Technology, Inc., #4812), rabbit polyclonal phosphorylated PKCβ (phospho-Thr-641) antibody (p-PKCβ; Signalway Antibody LLC, #11172), rabbit monoclonal IRF4 (D9P5H) antibody (Cell Signaling Technology, Inc., #15106), rabbit monoclonal BCL6 (D65C10) antibody (Cell Signaling Technology, Inc., #5650), rabbit monoclonal c-MYC (D84C12) antibody (Cell Signaling Technology, Inc., #5605), and rabbit monoclonal GAPDH (14C10) antibody (Cell Signaling Technology, Inc., #2118) were used.

Techniques: Activity Assay, Incubation, Inhibition, Cell Viability Assay, Western Blot

Journal: Brain

Article Title: Mutation in senataxin alters the mechanism of R-loop resolution in amyotrophic lateral sclerosis 4

doi: 10.1093/brain/awab464

Figure Lengend Snippet: ZPR1 co-localizes with SETX in nuclear bodies and its deficiency causes disruption of gems and Cajal bodies, downregulation of SETX and accumulation of R-loops . HeLa cells (Control) or transfected with 100 nM antisense oligonucleotides against human ZPR1 (As-ZPR1) or scrambled sequence oligo (Scramble) were fixed and stained with antibodies for immunofluorescence (IF) analysis. ( A ) Control and scramble oligo treated HeLa cells show ZPR1 (green) and SETX (red) co-localize in subnuclear foci (arrows) and knockdown of ZPR1 (As-ZPR1) causes disruption of SETX + foci and shows decrease in staining of SETX (red). ( B ) SETX (red) co-localizes with SMN (green) in nuclear gems (arrows) in control cells. As-ZPR1 causes disruption of SETX + (red) foci and SMN + (green) gems. ( C ) SETX (red) co-localizes with Coilin (green) in Cajal bodies (CBs) (arrows) in control cells. As-ZPR1 causes disruption of SETX + foci and Cajal bodies. ( D ) Quantification of SETX co-localization in subnuclear bodies (NBs)/cell (%) is shown as a violin plot with median and interquartile range (Q1, median, Q3) (50 cells/group). SETX co-localization with ZPR1: ZPR1 + SETX (72.73, 80.00, 100.00), SMN: SMN + SETX (32.89, 57.14, 80.83) and coilin: Coilin + SETX (24.31, 50.00. 68.75). Quantification of SETX co-localization (mean ± SEM, n = 50 cells/group) show the highest co-localization with ZPR1 (79.60 ± 3.03%) compared to SMN (54.01 ± 4.51%) (gems) and Coilin (47.00 ± 4.46%) (Cajal bodies). ( E ) Accumulation of RNA:DNA hybrids (R-loops) in ZPR1-deficient cells detected by monoclonal antibody (S9.6). As-ZPR1 causes accumulation of R-loops (green) and disruption of Cajal bodies (Coilin) (red). ( F ) ZPR1 (green) deficiency causes accumulation of γH2AX foci, a marker for DNA damage (red). ( G and H ) Specificity of R-loops detection by S9.6 antibody established by digestion of R-loops with the RNase H enzyme. Cells transfected with As-ZPR1 were permeabilized and treated with ( G ) buffer only and ( H ) RNase H enzyme for 20 min at room temperature, washed and fixed with 4% PFA. R-loops (green), coilin (red), nuclei (blue). Dotted circular lines indicate nuclei. Scale bar = 5.0 μm. ( I – K ) ZPR1 knockdown causes downregulation of SETX. ( I ) Immunoblots (IBs) of ZPR1, SETX, SMN and tubulin from cell lysates of Control, As-ZPR1 and Scramble transfected HeLa cells. Full-length blots are included in . ( J ) Quantitation of changes in ZPR1, SETX, and SMN protein levels with ZPR1 knockdown are shown as a scatter plot with median and range (min, median, max). ZPR1: Control (87.69, 95.35, 117.60), As-ZPR1 (18.36, 20.63, 28.32), Scramble (88.69, 90.32, 104.70); SETX: Control (82.79, 104.50, 113.70), As-ZPR1 (32.65, 40.36, 60.21), Scramble (90.65, 98.63, 111.70); SMN: Control (85.32, 96.32, 118.40), As-ZPR1 (37.65, 42.32, 50.32), Scramble (84.36, 105.40, 110.30). Quantitative (mean ± SEM, n = 3) and statistical analysis (ANOVA) show knockdown of ZPR1 levels to (22.44 ± 3.01%, P = 0.0002) decreases SETX levels to (44.41 ± 8.20%, P = 0.0036) and SMN levels to (43.43 ± 3.69%, P = 0.0026) compared to Control and Scramble. ( K ) Quantitation of changes in ZPR1, SETX, and SMN mRNA levels with ZPR1 knockdown are shown as a scatter plot with median and range. ZPR1: Control (93.70, 97.90, 109.90), As-ZPR1 (21.07, 35.09, 45.98), Scramble (84.65, 90.02, 109.70); SETX: Control (92.50, 95.50, 112.00), As-ZPR1 (30.50, 48.60, 64.17), Scramble (91.20, 95.20, 120.00); SMN: Control (95.70, 99.60. 107.20), As-ZPR1 (43.60, 49.00, 56.40), Scramble (94.87, 99.30, 108.00). Knockdown of ZPR1 mRNA expression to (34.05 ± 7.21%, P = 0.0007) causes downregulation of SETX mRNA expression to (47.76 ± 9.72%, P = 0.0061) and SMN mRNA to (49.67 ± 3.71%, P < 0.0001) compared to Control and Scramble. ( L ) Quantitative analysis of nuclear R-loop immunofluorescence intensity with NIH ImageJ software show ZPR1-deficient cells (As-ZPR1) accumulate R-loops (7.80 ± 0.37-fold, P < 0.0001) compared to Control and Scramble cells. R-loops nuclear intensity levels were quantified from three experiments (30 cells/group). Quantitative analysis of R-loop levels is shown as a scatter plot with median and range. Control (0.94, 0.97, 1.17), As-ZPR1 (7.29, 7.56, 8.54), Scramble (0.88, 0.98, 1.13). ( M ) Dot-blot analysis of R-loops using S9.6 antibody and genomic DNA isolated from control, As-ZPR1 and Scramble treated cells. ( N ) Densitometric quantitative analysis of R-loop levels in dot-blot shown as a scatter plot with median and range. Control (0.93, 0.99, 1.06), As-ZPR1 (6.81, 7.13, 7.91), Scramble (0.68, 1.04, 1.39). Quantitation of R-loop levels in dot-blot shows ZPR1-deficient cells (As-ZPR1) accumulate R-loops (7.28 ± 0.32-fold, P < 0.0001) compared to control and scramble cells. ( O and P ) Quantitative mapping of R-loop accumulation throughout transcription of the β-Actin ( ACTB ) and GAPDH genes. DRIP was performed using S9.6 antibody and genomic DNA prepared from control, control + RNase H, ZPR1-deficient (As-ZPR1) and As-ZPR1 + RNase H treated HeLa cells. DRIP and input DNA were used for qPCR analysis using specific primers pairs to amplify different regions of R-loop accumulation during transcription of the ACTB gene in ( O ) control, control + RNase H, As-ZPR1 and As-ZPR1 + RNase H, and ( P ) the GAPDH gene in control, control + RNase H, As-ZPR1 and As-ZPR1 + RNase H. Quantitative analysis (mean ± SEM, n = 3) shows ZPR1-deficiency causes ∼4-5-fold R-loop accumulation throughout transcription, including transcription start. Loss of R-loops with RNase H treatment shows specificity of DRIP analysis.

Article Snippet: Cells were blocked with 3% bovine serum albumin (BSA) in PBS with 0.5% Tween 20 (PBS-T) for 30 min at room temperature., Cells were double-labelled by sequential incubation with primary antibody against ZPR1 (clone LG1) or SMN (clone 8) or RNA:DNA hybrids (R-loops) (S9.6 antibody purified from hybridoma HB-8730, ATCC) for 1 h, washed 3× with PBS-T for 5 min each and incubated with secondary antibody Alexa488-conjugated anti-mouse IgG, washed 3× with PBS-T for 5 min each and followed by staining with second primary antibody against p80 Coilin (rabbit polyclonal, NBP2-15939, Novus Biologicals and mouse monoclonal; Clone 56; BD Biosciences; 612074) or DNA damage response (DDR) markers, γH2AX (rabbit NB100-79967, Novas Biologicals and mouse ab26350, Abcam), 53BP1 (4937, Cell Signaling), or SETX (ab220827, Abcam), p-DNA-PKcs (Ser2056) (ab18192), anti-β-tubulin class-III neuron-specific antibody (clone TUJ1, MAB1195, R & D Systems) for 1 h, washed 3× with PBS-T for 5 min, incubated with secondary antibody Alexa594-conjugated anti-rabbit IgG, washed 3× with PBS-T for 5 min. For ribonuclease H (RNase H) enzyme treatment, control and As-ZPR1 transfected cells were extracted with CSK buffer containing 0.1% Triton X-100 for 5 min followed by washing with CSK buffer without Triton-X100.

Techniques: Disruption, Transfection, Sequencing, Staining, Immunofluorescence, Marker, Western Blot, Quantitation Assay, Expressing, Software, Dot Blot, Isolation

Journal: Brain

Article Title: Mutation in senataxin alters the mechanism of R-loop resolution in amyotrophic lateral sclerosis 4

doi: 10.1093/brain/awab464

Figure Lengend Snippet: ZPR1 overexpression in vivo rescues DNA damage associated with R-loop accumulation and prevents degeneration of motor neurons in SMA . Primary spinal cord neurons were cultured from 7-day-old normal, SMA and Z-SMA (SMA mice with ZPR1 overexpression under the control of mouse Rosa26 promoter) mice. Neurons were differentiated in vitro for 12 days and stained with antibodies against neuron-specific β-tubulin-III (red), SMN, SETX, p-DNA-PKcs, R-loops and γH2AX, and immunofluorescence was examined by confocal microscopy. ( A – F ) Axonal defects include retraction, bending, folding of axons (arrowheads) that indicate degeneration of SMN-deficient neurons. ( A ) Staining of neurons with ZPR1 (green) and β-tubulin (red), ( B ) staining of neurons with SMN (green) and β-tubulin (red), ( C ) SETX (green) and β-tubulin (red), ( D ) p-DNA-PKcs (green) and β-tubulin (red), ( E ) R-loops (green) and β-tubulin (red) and ( F ) γH2AX (green) and β-tubulin (red). Insets show higher magnification of punctate staining of γH2AX foci indicating DNA damage. Z-SMA neurons with in vivo ZPR1 overexpression show rescue of degenerative features. Nuclei were stained with DAPI (blue). Scale bar = 25 μm. ( G ) Immunoblot analysis of cultured primary spinal cord motor neurons from Normal, SMA and Z-SMA mice for detecting changes in levels of ZPR1, SMN, SETX, DNA-PKcs, p-DNA-PKcs, and DNA damage marker, γH2AX (full-length blots, ). ( H ) Quantitation of protein levels in motor neurons from Normal, SMA, and Z-SMA mice is shown as a scatter plot with median and range (min, median, max). SMN: Normal (90.54, 95.64, 115.90), SMA (20.36, 25.87, 29.54), Z-SMA (70.65, 75.32, 85.65); ZPR1: Normal (90.21, 97.25, 112.50), SMA (44.25, 54.21, 55.47), Z-SMA (150.20, 170.30, 190.30); SETX: Normal (90.65, 98.54, 112.50), SMA (30.25, 39.87, 44.56), Z-SMA (70.25, 81.25, 94.21); p-DNA-PKcs: Normal (90.54, 100.30, 110.30), SMA (30.21, 37.32, 40.25), Z-SMA (70.25, 81.25, 94.21); DNA-PKcs: Normal (92.36, 97.25, 112.00), SMA (29.87, 32.65, 35.68), Z-SMA (75.32, 87.98, 100.70); γH2AX: Normal (90.25, 104,50, 108.0), SMA (287.5, 310.30, 322.60), Z-SMA (110.20, 120.40, 130.30). Statistical analysis (ANOVA) of immunoblot data (mean ± SEM, n = 3 mice/group) from spinal cord neurons shows increase in ZPR1 levels (1.70 ± 0.11-fold, P = 0.0001) results in increase of SMN levels to (77.21 ± 4.43%, P = 0.0002), SETX (88.73 ± 9.36%, P = 0.0012), p-DNA-PKcs (81.90 ± 6.92%, P = 0.0037) and total DNA-PKcs (87.98 ± 7.31%, P = 0.0018) leading to a marked decrease in γH2AX levels from 306.80 ± 10.26% to 120.30 ± 5.77% ( P = 0.0002). ( I ) Higher magnification images of nuclei of neurons stained with antibody against R-loops (green) and β-tubulin (red) from Normal, SMA and Z-SMA spinal cord neurons (dotted lines show nuclei). ( J ) Quantitation of nuclear immunofluorescence R-loop intensity in Normal, SMA, and Z-SMA spinal cord neurons is shown as a scatter plot with median and range. Normal (0.87, 0.95, 1.17), SMA (7.2, 8.25, 10.25), ZSMA (1.20, 1.60, 2.65). Quantitative analysis of nuclear R-loop immunofluorescence shows marked reduction in R-loop accumulation (1.82 ± 0.43-fold) in Z-SMA compared to SMA (8.58 ± 0.88)-fold ( P = 0.0023, t-test ), and compared to Normal (1.04 ± 0.09-fold) ( P = 0.0002, ANOVA) neurons.

Article Snippet: Cells were blocked with 3% bovine serum albumin (BSA) in PBS with 0.5% Tween 20 (PBS-T) for 30 min at room temperature., Cells were double-labelled by sequential incubation with primary antibody against ZPR1 (clone LG1) or SMN (clone 8) or RNA:DNA hybrids (R-loops) (S9.6 antibody purified from hybridoma HB-8730, ATCC) for 1 h, washed 3× with PBS-T for 5 min each and incubated with secondary antibody Alexa488-conjugated anti-mouse IgG, washed 3× with PBS-T for 5 min each and followed by staining with second primary antibody against p80 Coilin (rabbit polyclonal, NBP2-15939, Novus Biologicals and mouse monoclonal; Clone 56; BD Biosciences; 612074) or DNA damage response (DDR) markers, γH2AX (rabbit NB100-79967, Novas Biologicals and mouse ab26350, Abcam), 53BP1 (4937, Cell Signaling), or SETX (ab220827, Abcam), p-DNA-PKcs (Ser2056) (ab18192), anti-β-tubulin class-III neuron-specific antibody (clone TUJ1, MAB1195, R & D Systems) for 1 h, washed 3× with PBS-T for 5 min, incubated with secondary antibody Alexa594-conjugated anti-rabbit IgG, washed 3× with PBS-T for 5 min. For ribonuclease H (RNase H) enzyme treatment, control and As-ZPR1 transfected cells were extracted with CSK buffer containing 0.1% Triton X-100 for 5 min followed by washing with CSK buffer without Triton-X100.

Techniques: Over Expression, In Vivo, Cell Culture, In Vitro, Staining, Immunofluorescence, Confocal Microscopy, Western Blot, Marker, Quantitation Assay

Journal: Brain

Article Title: Mutation in senataxin alters the mechanism of R-loop resolution in amyotrophic lateral sclerosis 4

doi: 10.1093/brain/awab464

Figure Lengend Snippet: Modulation of ZPR1 levels regulates R-loop accumulation and rescues pathogenic R-loop phenotype in ALS4 patient cells. ( A – D ) Knockdown of ZPR1 in ALS4 patient cells increases R-loop accumulation and causes activation of DNA damage response. ALS4 cells were untransfected (Control) or transfected with (100 nM) antisense oligos (As-ZPR1) or scrambled sequence oligo (Scramble) (100 nM). Representative images are presented for double-labelled immunostainings: ( A ) ALS4 #3 patient cells stained for R-loops (green) and 53BP1 (red) and ( B ) ALS4 #4 patient cells stained for R-loops (green) and 53BP1 (red) show marked increase in R-loop accumulation in ZPR1-deficient ALS4 patient cells (As-ZPR1) compared to control and scramble oligo treated cells, which causes DNA damage response and accumulation of 53BP1. ZPR1-deficiency causes downregulation of SETX and DNA damage. ( C ) ALS4 #3 patient cells stained for SETX (red) and γH2AX (green) and ( D ) ALS4 #4 patient cells stained for SETX (red) and γH2AX (green) show marked increase γH2AX foci in the nucleus of ZPR1-deficient ALS4 patient cells (As-ZPR1) compared to control and scramble oligo treated cells. Scale bar = 5.0 μm. Quantitation (mean ± SEM, n = 3) of immunoblots from ALS4 #3 ( E, G ) and ALS4 #4 ( F and H ) control, As-ZPR1 and scramble samples show KD of ZPR1 to (19.69 ± 2.13%, P = 0.0001) in ALS4 #3 and (21.7 ± 4.614%, P < 0.0001) in ALS4 #4. ZPR1 KD decreases SETX level to (52.06 ± 6.55%, P = 0.0012) in ALS4 #3 and (53.28 ± 4.33%, P = 0.0002) in ALS4 #4. ( G ) Quantitation of changes in ZPR1 and SETX levels with ZPR1 knockdown in ALS4 #3 patient cells is shown as a scatter plot with median and range (min, median, max). ZPR1: ALS4 #3 Control (90.32, 103.30, 106.60), ALS4 #3 – As-ZPR1 (15.64, 20.54, 22.89), ALS4 #3 – Scramble (85.24, 98.54, 117.3); SETX: ALS4 #3 Control (92.21, 100.30, 108.60), ALS4 #3 – As-ZPR1 (40.56, 52.36, 63.25), ALS4 #3 – Scramble (93.68, 95.47, 109.60). ( H ) Quantitation of changes in ZPR1 and SETX levels with ZPR1 knockdown in ALS4 #4 patient cells is shown as a scatter plot with median and range. ZPR1: ALS4 #4 Control (92.31, 98.32, 112.40), ALS4 #4 – As-ZPR1 (12.54, 25.31, 27.25), ALS4 #4 – Scramble (94.32, 99.65, 105.3); SETX: ALS4 #4 Control (95.45, 98.24, 108.6), ALS4 #4 – As-ZPR1 (45.32, 54.32, 60.21), ALS4 #4 – Scramble (94.32, 98.32, 105.3). ( I and J ) Overexpression of ZPR1 in ALS4 patient cells rescues disease phenotype and improves accumulation of R-loops. ( I ) Control experiment with Normal and ALS4 patient cells infected with adenovirus Ad5-GFP (100 MOI) expressing GFP (green) and stained with antibody against R-loops (red) did not show any change in the normal levels of R-loop fluorescence intensity in the nucleus. ( J ) Normal and ALS4 cells infected with Ad5-ZPR1-GFP expressing ZPR1-GFP (green) and stained for R-loops (red) show that ZPR1 overexpression decreases R-loop accumulation in the Normal (arrows) and rescues R-loop accumulation in ALS4 patient cells (arrows). Nuclei were stained with DAPI (blue). Scale bar = 20 μm. Arrows show infected cells and asterisks indicate non-infected cells. Dotted circular lines indicate nuclei. ( K – N ) ZPR1 overexpression improves in vivo association of SETX with R-loops in ALS4 patient cells. ( K ) Immunoblots of ZPR1-GFP, SETX, GFP and tubulin proteins in Normal and ALS4 patient cells overexpressing GFP and ZPR1-GFP. ( L ) Quantitation of SETX levels in Normal and ALS4 patient cells overexpressing GFP and ZPR1-GFP is presented as a scatter plot with median and range. Normal-GFP (94.65, 98.36, 108.70); Normal-ZPR1-GFP (187.40, 210.40, 250.00); ALS4-GFP (87.65, 92.35, 110.30); ALS4-ZPR1-GFP (169.40, 197.30, 240.30). ( M ) Immunoprecipitation of R-loops from Normal and ALS4 cells overexpressing GFP and ZPR1-GFP shows increase in in vivo binding of SETX with R-loops. ( N ) Quantitation of SETX protein levels co-IP with R-loops in Normal and ALS4 patient cells overexpressing GFP and ZPR1-GFP is presented as a scatter plot with median and range. Normal-GFP (90.76, 102.50, 107.90); Normal-ZPR1-GFP (115.60, 147.00, 160.30); ALS4-GFP (25.98, 36.10, 40.53); ALS4-ZPR1-GFP (74.98, 82.76, 112.40). Quantitation and comparison of SETX levels in co-IP with R-loops show increase in SETX binding with R-loops (90.03 ± 11.39%) compared to (34.24 ± 4.30%, P = 0.0101) in ALS4 expressing ZPR1-GFP and GFP, respectively. All full-length blots are included in . Enlarged images for ( I ) and ( J ) are included in .

Article Snippet: Cells were blocked with 3% bovine serum albumin (BSA) in PBS with 0.5% Tween 20 (PBS-T) for 30 min at room temperature., Cells were double-labelled by sequential incubation with primary antibody against ZPR1 (clone LG1) or SMN (clone 8) or RNA:DNA hybrids (R-loops) (S9.6 antibody purified from hybridoma HB-8730, ATCC) for 1 h, washed 3× with PBS-T for 5 min each and incubated with secondary antibody Alexa488-conjugated anti-mouse IgG, washed 3× with PBS-T for 5 min each and followed by staining with second primary antibody against p80 Coilin (rabbit polyclonal, NBP2-15939, Novus Biologicals and mouse monoclonal; Clone 56; BD Biosciences; 612074) or DNA damage response (DDR) markers, γH2AX (rabbit NB100-79967, Novas Biologicals and mouse ab26350, Abcam), 53BP1 (4937, Cell Signaling), or SETX (ab220827, Abcam), p-DNA-PKcs (Ser2056) (ab18192), anti-β-tubulin class-III neuron-specific antibody (clone TUJ1, MAB1195, R & D Systems) for 1 h, washed 3× with PBS-T for 5 min, incubated with secondary antibody Alexa594-conjugated anti-rabbit IgG, washed 3× with PBS-T for 5 min. For ribonuclease H (RNase H) enzyme treatment, control and As-ZPR1 transfected cells were extracted with CSK buffer containing 0.1% Triton X-100 for 5 min followed by washing with CSK buffer without Triton-X100.

Techniques: Activation Assay, Transfection, Sequencing, Staining, Quantitation Assay, Western Blot, Over Expression, Infection, Expressing, Fluorescence, In Vivo, Immunoprecipitation, Binding Assay, Co-Immunoprecipitation Assay, Comparison

Journal: Scientific Reports

Article Title: Acute ischemic stroke triggers a cellular senescence-associated secretory phenotype

doi: 10.1038/s41598-021-95344-5

Figure Lengend Snippet: Microglial cells of the ischemic area showed a higher expression of NF-kB and γ-H2AX. ( A ) Immunofluorescence NF-kB expression showed an infarct area location, delimited by white dotted line. Plot represented the NF-kB expression by areas (contralateral vs infarct area: p < 0.0001; ipsilateral vs infarct: p = 0.0009; contralateral vs ipsilateral: p < 0.0001). Scale bar = 100 µm; insert scale bar = 40 µm. ( B ) Nuclear expression of γ-H2AX in microglial cells. Plot represented the γ-H2AX positive foci in contralateral, ipsilateral and infarct areas (contralateral vs infarct: p < 0.0001; ipsilateral vs infarct: p = 0.0004). Scale bar = 100 µm; insert scale bar = 50 µm.

Article Snippet: Immunohistochemistry was performed against mouse anti-p16 (1:200; Abcam, Cat. No. ab54210), mouse anti-p21 (1:200; Santa Cruz Biotechnology, Cat. No. sc-817), rabbit anti-GFAP (1:200; Abcam, Cat. No. ab7260), rabbit anti-Iba1 (1:200; FUJIFILM Wako, Cat. No. 019-19741), rabbit anti-NeuN (1:200; Abcam, Cat.No.ab177487), rabbit anti-NF-κB p65 (1:200; Proteintech, Cat. No. 10745-1-AP), chicken anti-Iba1 (1:200; Synaptic Systems, Cat No. 234 006), and rabbit gamma H2AX [p Ser139] antibody (EP854(2)Y, 1:200; NOVUS Biologicals, Cat. No. NB100-79967) primary antibodies and followed by appropriated secondary antibodies.

Techniques: Expressing, Immunofluorescence

Journal: Frontiers in Public Health

Article Title: The Use of Genotoxicity Endpoints as Biomarkers of Low Dose Radiation Exposure in Interventional Cardiology

doi: 10.3389/fpubh.2021.701878

Figure Lengend Snippet: Mean values of chromosomal aberrations, frequency of Micronuclei (MN), and γ-H2AX foci per cell before the exposure, immediately after the interventional procedure ( in vivo radiation), and 24 h later, as well as the percentage of γ-H2AX foci repaired at 24 h.

Article Snippet: The main steps were the permeabilization of the cells, blocking of non-specific binding, immunostaining with primary γ-H2AX antibody (rabbit 1:1,000, Cat: NB100-79967, Novus Biologicals, Abingdon, UK) and secondary fluorescent antibody (Rhodamine Red-X anti-rabbit, 1:4,000, Cat: R6394, Life Technologies).

Techniques: In Vivo

Journal: Frontiers in Public Health

Article Title: The Use of Genotoxicity Endpoints as Biomarkers of Low Dose Radiation Exposure in Interventional Cardiology

doi: 10.3389/fpubh.2021.701878

Figure Lengend Snippet: Mean values of γ-H2AX foci and frequency of Micronuclei (MN) per cell after cardiac interventional procedures ( in vivo radiation) and after irradiation with 1 Gy in the laboratory ( in vitro radiation).

Article Snippet: The main steps were the permeabilization of the cells, blocking of non-specific binding, immunostaining with primary γ-H2AX antibody (rabbit 1:1,000, Cat: NB100-79967, Novus Biologicals, Abingdon, UK) and secondary fluorescent antibody (Rhodamine Red-X anti-rabbit, 1:4,000, Cat: R6394, Life Technologies).

Techniques: In Vivo, Irradiation, In Vitro