Journal: Journal of Leukocyte Biology

Article Title: Retinoblastoma protein induction by HIV viremia or CCR5 in monocytes exposed to HIV-1 mediates protection from activation-induced apoptosis: ex vivo and in vitro study

doi: 10.1189/jlb.1111552

Figure Lengend Snippet: (A) Monocytes obtained from HIV− (n=16) and HIV+ (n=15) and suppressed (n=24) donors incubated with 20 μM CdCl2 overnight (16 h). Apoptosis induction was determined by using flow cytometry to detect active caspase-3. p53ser15 (B), Rb1 (C), and p21 (D) expression in CD14+ monocytes from HIV− donors (n=12) as compared with HIV+ donors (n=14) and suppressed donors (n=23). MFI, Mean fluorescence intensity. (E) Scatter plot images with Rb1 expression levels in CD14+ cells are shown plotted against each donor's log10 VL. Rb1 levels correlated positively with VL. Pearson's correlation coefficient and P value are listed. Monocytes were enriched from PBMCs from HIV− (C; n=6) and HIV+ donors (P; n=6) and lysed, and PUMA (F) and PAI-1 (G) were detected by Western blotting. Data shown are representative of six viremic donors tested. *denotes P < 0.05.

Article Snippet: Cells were incubated at 4°C for 30 min. Precleared lysates were mixed with 4× SDS loading buffer containing 1 μM DTT and heated to 100°C for 5 min. Lysates were resolved on precast 4–15% Tris-HCl gels (Bio-Rad Laboratories), and proteins were subsequently transferred to PVDF membranes (Bio-Rad Laboratories), blocked with 1% BSA in PBS with 0.02% v/v Tween-20, and detected with primary antibodies: goat anti-human p21 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human Rb1 (Santa Cruz Biotechnology), mouse anti-human PAI-1 (BD Biosciences), rabbit polyclonal to PUMA (Abcam, Cambridge, MA, USA), LC3A/B antibody (Cell Signaling Technology), or rabbit polyclonal to Beclin (Cell Signaling Technology).

Techniques: Incubation, Flow Cytometry, Expressing, Fluorescence, Western Blot

Journal: Journal of Leukocyte Biology

Article Title: Retinoblastoma protein induction by HIV viremia or CCR5 in monocytes exposed to HIV-1 mediates protection from activation-induced apoptosis: ex vivo and in vitro study

doi: 10.1189/jlb.1111552

Figure Lengend Snippet: Monocytes were incubated in the presence or absence of 50 ng/ml R5 HIV-1 or 1 μg/ml MIP-1β and harvested after 36 h for real-time RT-PCR analysis of total RNA performed with p53-specific (A) and Rb1-specific (B) primers, and data were normalized to β-actin. The experiment was repeated three times. Whole cell lysates were prepared and analyzed by SDS-PAGE, followed by immunoblotting with anti-Rb1 and anti-p21 antibody (C). (D) Monocytes were pretreated with (right panel) or without (left panel) 15 μM Maraviroc (MRV) and then exposed to R5 HIV-1. p21 expression was detected after 36 h and analyzed by flow cytometry (data representative of two separate experiments). (E) Cell lysates prepared as in C but probed with anti-LC3A/B antibody (left panel) or anti-Beclin antibody (right panel). β-Actin was used as a protein loading control. The experiment is repeated in three additional donors. *P < 0.05 (see brackets).

Article Snippet: Cells were incubated at 4°C for 30 min. Precleared lysates were mixed with 4× SDS loading buffer containing 1 μM DTT and heated to 100°C for 5 min. Lysates were resolved on precast 4–15% Tris-HCl gels (Bio-Rad Laboratories), and proteins were subsequently transferred to PVDF membranes (Bio-Rad Laboratories), blocked with 1% BSA in PBS with 0.02% v/v Tween-20, and detected with primary antibodies: goat anti-human p21 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human Rb1 (Santa Cruz Biotechnology), mouse anti-human PAI-1 (BD Biosciences), rabbit polyclonal to PUMA (Abcam, Cambridge, MA, USA), LC3A/B antibody (Cell Signaling Technology), or rabbit polyclonal to Beclin (Cell Signaling Technology).

Techniques: Incubation, Quantitative RT-PCR, SDS Page, Western Blot, Expressing, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: Retinoblastoma protein induction by HIV viremia or CCR5 in monocytes exposed to HIV-1 mediates protection from activation-induced apoptosis: ex vivo and in vitro study

doi: 10.1189/jlb.1111552

Figure Lengend Snippet: Monocytes from uninfected donors were treated with negative-control siRNA or Rb1 siRNA. Knockdown efficiency was confirmed by real-time RT-PCR and Western blot (A). (B) Cells with target or control siRNAs against Rb1 were then incubated in the absence (upper panels) or presence (lower panels) of R5 HIV-1 and subsequently challenged with 100 μM CdCl2 for 5 h. Apoptosis induction was determined by detecting active caspase 3 by flow cytometry. Results were reproduced in monocytes from five different donors (C). (D) Cells with siRNAs against TP53 (left) or treated in the absence or presence of PFT-α (right) were incubated with (lower panels) or without (upper panels) R5 HIV-1. Apoptosis induction was determined by detecting active caspase 3 by flow cytometry.

Article Snippet: Cells were incubated at 4°C for 30 min. Precleared lysates were mixed with 4× SDS loading buffer containing 1 μM DTT and heated to 100°C for 5 min. Lysates were resolved on precast 4–15% Tris-HCl gels (Bio-Rad Laboratories), and proteins were subsequently transferred to PVDF membranes (Bio-Rad Laboratories), blocked with 1% BSA in PBS with 0.02% v/v Tween-20, and detected with primary antibodies: goat anti-human p21 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human Rb1 (Santa Cruz Biotechnology), mouse anti-human PAI-1 (BD Biosciences), rabbit polyclonal to PUMA (Abcam, Cambridge, MA, USA), LC3A/B antibody (Cell Signaling Technology), or rabbit polyclonal to Beclin (Cell Signaling Technology).

Techniques: Negative Control, Quantitative RT-PCR, Western Blot, Incubation, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: Retinoblastoma protein induction by HIV viremia or CCR5 in monocytes exposed to HIV-1 mediates protection from activation-induced apoptosis: ex vivo and in vitro study

doi: 10.1189/jlb.1111552

Figure Lengend Snippet: Figure illustrating the possible relationship between HIV binding to monocyte surface and the functional outcome associated with Rb1 activation.

Article Snippet: Cells were incubated at 4°C for 30 min. Precleared lysates were mixed with 4× SDS loading buffer containing 1 μM DTT and heated to 100°C for 5 min. Lysates were resolved on precast 4–15% Tris-HCl gels (Bio-Rad Laboratories), and proteins were subsequently transferred to PVDF membranes (Bio-Rad Laboratories), blocked with 1% BSA in PBS with 0.02% v/v Tween-20, and detected with primary antibodies: goat anti-human p21 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human Rb1 (Santa Cruz Biotechnology), mouse anti-human PAI-1 (BD Biosciences), rabbit polyclonal to PUMA (Abcam, Cambridge, MA, USA), LC3A/B antibody (Cell Signaling Technology), or rabbit polyclonal to Beclin (Cell Signaling Technology).

Techniques: Binding Assay, Functional Assay, Activation Assay

Journal: Cellular and Molecular Life Sciences

Article Title: NPC transplantation rescues sci-driven cAMP/EPAC2 alterations, leading to neuroprotection and microglial modulation

doi: 10.1007/s00018-022-04494-w

Figure Lengend Snippet: Effects of dual NPC and ESI-05 treatment on scar area, microglial phenotype, and NeuN + cell gap. A Quantification of the scar area (GFAP-) throughout longitudinal slices in the mid-region of the injured spinal cord. Two-way ANOVA: **** p < 0.0001. B Representative images of GFAP immunostaining and traced GFAP- area (white line). C Morphological visualization of microglial cells by Iba-1 staining and determination of the percentage of ramified microglial cells. D Pie charts representing the microglial population segregated in ramified or circular cells for each experimental group. E Representative images of P2X4 immunostaining and its quantification. One-way ANOVA: * p < 0.05. F Representative images (left panel) of the sparsity of NeuN + cells through the injury and quantification of the gap distance (right panel). G Heat map showing the mean number of NeuN + cells per experimental group in each spinal segment of 2 mm

Article Snippet: Primary antibodies used were—anti-B-III-Tubulin (α-ms,1:400, MO15013, Neuromics, Edina, MN, USA), anti-NeuN (α-CK,1:600, ABN91, Merck Millipore, Massachusetts, USA), anti-GFAP (α-CK, 1:1000 PA1-10,004, Thermo Fisher, Massachusetts, USA), anti-cAMP (α-Rb,1:100, 07–1497, Merck Millipore, Massachusetts, USA), anti-Epac2 (α-Rb,1:400, 43,239, Cell Signalling Technology, Massachusetts, USA), Iba-1 (α-Rb,1:400, 019–19,741, WAKO, Osaka, Japan), P2X4 (α-Rb, 1:400, purchased from Alomone Labs, Jerusalem, Israel).

Techniques: Immunostaining, Staining

Journal: BMC Biotechnology

Article Title: Simultaneous silencing of multiple RB and p53 pathway members induces cell cycle reentry in intact human pancreatic islets

doi: 10.1186/1472-6750-14-86

Figure Lengend Snippet: Simultaneous knockdown of multiple genes in primary human islets following electroporation. (A) Graph shows RT-qPCR results of islets electroporated with non-targeting control siRNA or siRNA targeting RB and p130 (L-003299). (B) Graph shows RT-qPCR results of islets electroporated with non-targeted control siRNA or siRNAs targeting the indicated gene products. The housekeeping gene GAPDH was used as the control. Representative experiment shown from 3 separate experiments. Error bars indicate standard deviation. (C) Western blot for RB protein in human islet lysates after electroporation of control or targeting siRNA. GAPDH was used as a loading control. RB appears as a characteristic doublet representing the hypo and hyperphosphorylated protein. Representative experiment shown from 3 different experiments.

Article Snippet: For detection of Retinoblastoma protein blots were probed with 0.1 μg/mL of Human RB1 (MAB6495, R&D Systems) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (R &D Systems).

Techniques: Electroporation, Quantitative RT-PCR, Standard Deviation, Western Blot