Journal: Cells

Article Title: New Insights into the Significance of PARP-1 Activation: Flow Cytometric Detection of Poly(ADP-Ribose) as a Marker of Bovine Intramammary Infection

doi: 10.3390/cells10030599

Figure Lengend Snippet: Response to 16 h LPS treatment of bovine PBMC. ( A ) Representative histograms overlay of PAR content in control (black line) and LPS treated PBMC (green line). ( B ) Histogram showing the decrease in MFI PAR (Geometric mean) in PBMC in response to 16h LPS treatment ( p = 0.048). ( C ) Histogram overlay showing the amount of active caspase-3 positive PBMC; ( D ) histogram overlay showing the amount of cleaved PARP-1 positive PBMC. ( E ) Percentage of active Caspase-3 positive cells in control and LPS treated PBMC. ( F ) Percentage of cleaved-PARP-1 positive cells in control and LPS treated PBMC. Results are presented as mean ± SD of n = 3 cows. * Significant differences with p < 0.05, paired Student’s t -test.

Article Snippet: In order to verify apoptotic inactivation of PARP after prolonged LPS stimulation, we evaluated the activation of Caspase-3 and the cleavage of PARP-1 by using the PE- conjugated anti-active Caspase-3 (clone C92-605—Becton Dickinson) and the FITC- conjugated anti-cleaved PARP-1 (Asp214) (clone F21-852, BD—Becton Dickinson) monoclonal antibodies, respectively.

Techniques:

Journal: Cells

Article Title: New Insights into the Significance of PARP-1 Activation: Flow Cytometric Detection of Poly(ADP-Ribose) as a Marker of Bovine Intramammary Infection

doi: 10.3390/cells10030599

Figure Lengend Snippet: Flow cytometric evaluation of PAR in bovine milk leukocytes and PARP-1 activation in milk cells during infection. At the top, representative flow cytometry dot plots showing the gating strategy for the identification of leukocyte subpopulations: ( A ) Identification of single cells on FSC-H vs. FSC-A dot-plot, ( B ) identification of milk leukocytes (CD45 + ) in SSC vs. PE dot-plot, ( C ) identification of lymphocytes (CD14 − ) neutrophils (CD14 low/− ) and macrophages (CD14 + ) in SSC vs. Per-CP dot-plot. In the middle, histograms overlay showing PAR levels of the three different cell populations: ( D ) Lymphocytes, ( E ) macrophages, and ( F ) neutrophils. Below, box-whisker plots (median, 25th–75th percentiles, maximum and minimum values) showing the PAR MFI differences of the leukocyte subpopulations between not infected (NI, n = 9) and infected (I, n = 10) milk samples ( G ), lymphocytes; ( H ), macrophages, ( I ), neutrophils). A significant increase in PAR levels was observed in lymphocytes ( p = 0.02) and macrophages ( p < 0.01) in infected conditions after the unpaired Student’s t -test. * Significant differences with p < 0.05.

Article Snippet: In order to verify apoptotic inactivation of PARP after prolonged LPS stimulation, we evaluated the activation of Caspase-3 and the cleavage of PARP-1 by using the PE- conjugated anti-active Caspase-3 (clone C92-605—Becton Dickinson) and the FITC- conjugated anti-cleaved PARP-1 (Asp214) (clone F21-852, BD—Becton Dickinson) monoclonal antibodies, respectively.

Techniques: Activation Assay, Infection, Flow Cytometry, Whisker Assay

Journal: RSC Advances

Article Title: Apoptosis induction and cell cycle arrest induced by Sinkiangenone B, a novel phenylpropanoid derivative from the resin of Ferula sinkiangensis K. M. Shen

doi: 10.1039/c7ra13716h

Figure Lengend Snippet: Fig. 8 The effects of Sinkiangenone B on the expression of apoptosis-related proteins determined by western blot. AGS cells were treated with Sinkiangenone B (0, 5, 10, 15 mm) for 24 h. Sinkiangenone B decreased the expression of Bcl-2 and cleaved PARP, and increased the expression of Bax and cleaved caspase-3. Relative expression levels of apoptosis-related proteins were showed. b-Tubulin was used to confirm equal protein loading. *p < 0.05 and **p < 0.01 were considered statistically significant.

Article Snippet: Antibodies against Bax, Bcl-2, cleaved PARP, cleaved caspase-3, cyclin D1, cyclin E, Cdk4, Cdk2, P16, P27 and P53 were purchased from Santa Cruz Biotechnology RSC Adv., 2018, 8, 4093–4103 | 4101 O pe n A cc es s A rt ic le .

Techniques: Expressing, Western Blot

Journal: Cell reports

Article Title: Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N -Glycosylation of a Chaperone

doi: 10.1016/j.celrep.2020.107840

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used: HSP90 (SMC-107) from Stressmarq; HER2 (28–0004), myc (R950–25) from Invitrogen; Calnexin (610523) from BD Biosciences; HSP70 (SPA-810), GRP94 (SPA-850) from Enzo; GAPDH (ab8245), GRP78 (ab21685), HSP90α (ab2928) from Abcam; cleaved PARP (G7341) from Promega; EGFR (4267), LRP6 (2560), p-AKT (S473) (9271), AKT (4691), Caspase 7 (9494), p-ERK1/2 (T202/Y204) (4377), ERK1/2 (4695), p-STAT3 (9145), STAT3 (12640), p-p65 (S536) (3033), p65 (8242), Flotillin-1 (3253) from Cell Signaling Technology; GRP94 (G4420), a-Tubulin (T5168), b-actin (A1978) and Concanavalin A-HRP (L6397) from Sigma-Aldrich.

Techniques: Recombinant, Isolation, Cell Fractionation, Viability Assay, Transfection, Software, Western Blot, Staining

Journal: eLife

Article Title: A subset of CB002 xanthine analogs bypass p53-signaling to restore a p53 transcriptome and target an S-phase cell cycle checkpoint in tumors with mutated-p53

doi: 10.7554/eLife.70429

Figure Lengend Snippet: CB002 structural analogs activate p53 reporter gene activity in SW480 cells in a dose-dependent manner (6 hr) ( A ). Therapeutic indices for CB002-structural analogs were determined in SW480 cells (48 hr) ( B ). Propidium iodide cell cycle analysis was performed to determine sub-G1 population at 48 hr of treatment with CB002-analogs at 100 μM in SW480 cells. Two-way ANOVA, p<0.05 ( C ). CB002-analog #4 restores the p53 pathway in SW480 cells, resulting in PARP cleavage independently of p73 ( D ). Immunofluorescence staining of Cyt-C (green), Tom20 (red) DAPI (blue) in SW480 treated as indicated for 48 hr ( E ). Noxa protein expression induced by CB002-analogs in DLD-1, SW480, HCT116, and HCT116 p53 (R175H) colorectal cancer cells (24 hr) ( F ). p53-pathway restoring compounds have unique properties compared to other xanthine derivatives in their ability to induce Noxa expression, 24 hr treatment in DLD-1 cells ( G ). Xanthine derivatives CB002 and its analog induce Noxa expression but not caffeine, pentoxifylline, and theophylline at 24 hr in DLD-1 and SW480 cells ( H ). ATF3/4 mediate Noxa induction ( I ). Caffeine (C), Pentoxifylline (P), and Theophylline (T). Figures (A)–(C) were performed as three biological replicates. Experiments from figures (D)–(I) were performed at least twice and a representation of one is shown.

Article Snippet: The following antibodies were used: cytochrome C (1:1000; #sc-13560; Santa Cruz; RRID: AB_627383 ), p53 (1:1000; #sc-126; Santa Cruz; RRID: AB_628082 ), p73 (1:1000; #A300-126A; Bethyl Laboratories), Noxa (1:250; #OP180; EMD Millipore; RRID: AB_564933 ), DR5 (1:1,000; #3696; Cell Signaling Technology; RRID: AB_10692107 ), cleaved PARP (1:1,000; #9546; Cell Signaling Technology; RRID: AB_2160593 ), ATF3 (1:1000, #sc-188, Santa Cruz; RRID: AB_2258513 ), ATF4 (1:1,000; #11815; Cell Signaling Technology), p-RPA32/RPA2 (Ser8) (1:1,000; #54762, Cell Signaling Technology), RPA32/RPA2 (1:1000; 52448; Cell Signaling Technology), p-cdc2 (Tyr15) (1:1000; #9111; Cell Signaling Technology), cdc2 (1:1000; #54; Santa Cruz Biotechnology), p-cdc25c (Ser216) (1:1000; #9528; Cell Signaling Technology; RRID: AB_2075150 ), cdc25c (1:1000; #13138; Santa Cruz Biotechnology; RRID: AB_627227 ), p-H3 (Ser10) (1:1000; #3377; Cell Signaling Technology; RRID: AB_1549592 ), H3 (1:1000; #14269; Cell Signaling Technology; RRID: AB_2756816 ), γ-H2AX (Ser139) (1:1000; #2577; Cell Signaling Technology; RRID: AB_2118010 ), p-ATR (Thr1989) (1:1000; GTX128145, GeneTex; RRID: AB_2687562 ), ATR (1:1000; #1887; Santa Cruz Biotechnology; RRID: AB_630893 ), Cyclin A (1:1000; sc-271682, Santa Cruz Biotechnology;), p21 (1:200; #OP64; EMD Millipore; RRID: AB_2335868 ), Ran (1:10000; #610341; BD Biosciences; RRID: AB_397731 ), and β-actin (1:10000, A5441, Sigma-Aldrich; AB_476744).

Techniques: Activity Assay, Cell Cycle Assay, Immunofluorescence, Staining, Expressing