Journal: Nursing research

Article Title: Differential Gene Expression in Erlotinib-treated Fibroblasts

doi: 10.1097/NNR.0000000000000330

Figure Lengend Snippet: Confirmation that cultured cells (ATCC® PCS-201-012™) were fibroblasts and that treatment with the tyrosine kinase inhibitor erlotinib did not impinge upon cell viability. (A) Representative fluorescent micrographs show expression of indicated markers in fibroblasts. Cells were thawed, resuspended, and grown to 80% confluence per procedure, and then transferred to 25 mm coverslips (3 coverslips per condition). Cells were fixed and then immunolabeled with the primary antibody, anti-TE-7 (1:100). The secondary antibody was FITC conjugated donkey anti-mouse IgG (1:100). White arrows indicate cell bodies labeled with DAPI and anti-TE-7. Each experiment was performed in triplicate. (B) There was no statistical difference among the treatment groups (F2,6 = 0.088; p = 0.917). Treatment with the tyrosine kinase inhibitor erlotinib did not impinge upon cell viability (C). Human fibroblasts (ATCC® PCS-201-012™, Manassas, VA) were cultured in flat-bottomed 96-well plates according to standard protocol. Fibroblasts were treated with vehicle, staurosporine, and erlotinib (1–10μM) every day for three days. After three days, the effects of treatment on cell viability were assessed using the Vybrant® MTT Cell Proliferation Kit (V-13154, Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Absorbance of the solution was measured at 540 nM(1sec) to quantify the amount of formazan produced by the reaction. Cell viability was computed as mean percentage of control with the no treatment group designated as the reference group. Staurosporine served as a positive control for cell death. The mean of three individual experiments is represented. There was a statistical difference (F6,14 = 9.023; p < 0.0004) between no treatment and the staurosporine (*p < 0.0001) group and the no treatment and erlotinib 10μM (**p = 0.005) group.

Article Snippet: Methods: Dermal fibroblasts (ATCC® PCS-201-012TM) were seeded in cell culture flasks, grown under standard conditions, and transferred to cell culture dishes.

Techniques: Cell Culture, Expressing, Immunolabeling, Labeling, MTT Cell Proliferation, Produced, Control, Positive Control

Journal: Nursing research

Article Title: Differential Gene Expression in Erlotinib-treated Fibroblasts

doi: 10.1097/NNR.0000000000000330

Figure Lengend Snippet: Top 20 genes differentially regulated in fibroblasts treated with 1μM erlotinib versus control.

Article Snippet: Methods: Dermal fibroblasts (ATCC® PCS-201-012TM) were seeded in cell culture flasks, grown under standard conditions, and transferred to cell culture dishes.

Techniques: Control, Over Expression, Expressing, Clinical Proteomics, Membrane, Biomarker Discovery, Irradiation, Sequencing, Phospho-proteomics, Marker, Binding Assay, Blocking Assay, Activation Assay, Inhibition, Translocation Assay, Gene Expression, Functional Assay, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Activity Assay, Variant Assay

Journal: Nursing research

Article Title: Differential Gene Expression in Erlotinib-treated Fibroblasts

doi: 10.1097/NNR.0000000000000330

Figure Lengend Snippet: Validation of differentially expressed genes from microarray analysis using qPCR. Fresh fibroblasts were cultured and underwent experimental procedures as described. Results are reported as linear fold changes in erlotinib 1uM as compared to collapsed control; n = 3 per group for each microarray and qPCR. qPCR = quantitative real-time polymerase chain reaction. Solute carrier family 7 [anionic amino acid transporter light chain] member 7, = SLC7A11; Matrix metallopeptidase 12 = MMP12; chemokine (C-C motif) ligand 2 = CCL2; cell division cycle 6 homolog = CDC6; DIRAS3 = DIRAS family, GTP-binding RAS-like 3

Article Snippet: Methods: Dermal fibroblasts (ATCC® PCS-201-012TM) were seeded in cell culture flasks, grown under standard conditions, and transferred to cell culture dishes.

Techniques: Biomarker Discovery, Microarray, Cell Culture, Control, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Nursing research

Article Title: Differential Gene Expression in Erlotinib-treated Fibroblasts

doi: 10.1097/NNR.0000000000000330

Figure Lengend Snippet: Validation of gene expression analyses using Western blot analysis. Analysis of protein lysate from fibroblasts in the combined control group (no treatment, vehicle) and the erlotinib 1μM identified target protein level for MMP12, CCL2, CDC6, and SLC7A11. The protein level of MMP12 and SCL7A11 was upregulated 240%, and 262%, respectively, in the erlotinib 1μM condition compared to control. The protein level of CCL2 and CDC6 was downregulated 41% and 27%, respectively, in the in the erlotinib 1μM condition compared to control. Beta actin was used as the reference product. Solute carrier family 7 [anionic amino acid transporter light chain] member 7, = SLC7A11; Matrix metallopeptidase 12 = MMP12; chemokine (C-C motif) ligand 2 = CCL2; cell division cycle 6 homolog = CDC6.

Article Snippet: Methods: Dermal fibroblasts (ATCC® PCS-201-012TM) were seeded in cell culture flasks, grown under standard conditions, and transferred to cell culture dishes.

Techniques: Biomarker Discovery, Gene Expression, Western Blot, Control

Journal: Oncotarget

Article Title: Shh and p50/Bcl3 signaling crosstalk drives pathogenesis of BCCs in Gorlin syndrome.

doi: 10.18632/oncotarget.5103

Figure Lengend Snippet: Figure 3: Ionizing radiation (IR) induce multiple BCCs in Ptch1+/−/SKH-1 mice. A. Representative pictures of Ptch+/−/ SKH-1 mice showing IR-induced visible BCCs on dorsal and ventral skin. B. BCCs/ mouse. C. tumor volume/mouse; D. Histology of BCCs from dorsal and ventral skin, and analysis of microscopic BCCs/unit area (mm2) in IR-irradiated mice. E. Immunoblot analysis of biomarkers predictive of cell proliferation (PCNA and cyclin D1) and anti-apoptotic protein Bcl2 in BCCs of IR-irradiated mice. F. transcriptional expression of Glis, Ptchs and cell cycle regulatory cyclins in IR-irradiated BCC. Ptch+/−/SKH-1 mice were irradiated with a single dose (5Gy) of IR. The experiment was terminated at week 20 following irradiation. Skin and tumors were excised for histological and for molecular analysis.

Article Snippet: The non-specific sites were blocked in 2.5% goat serum and bovine serum albumin (0.5% w/v) for 1 hour followed by overnight incubation at 4°C in the following primary antibodies: PCNA (Santa Cruz, sc-9857, 1:100), Gli-1 (Santa Cruz, sc-20687, 1:100); Hhip (Santa Cruz, sc-9406, 1:100).

Techniques: Irradiation, Western Blot, Expressing

Journal: Oncogene

Article Title: AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

doi: 10.1038/s41388-019-0724-7

Figure Lengend Snippet: Miniaturized western blot setup to perform a screening of PCNA ubiquitylation inhibitors. a U2OS cells were UV irradiated (15 J/m 2 ) and treated for 12 h with the proteasome inhibitors Epoxomicin and MG-132. The western blot was performed with two monoclonal antibodies to simultaneously detect total PCNA (in red) and ubi-PCNA (in green) using a LI-COR Odyssey infrared scanner. The ratios of ubi-PCNA/total PCNA were normalized to the highest induction of ubi-PCNA in the non-treated (NT) UV-irradiated sample. b Three days detailed protocol to screen for PCNA ubiquitylation inhibitors, showing the quality controls to ensure reproducibility and robustness of PCNA ubiquitylation induction: (i) use of an infrared scanner to confirm the homogenous distribution of cells in the wells across the entire plate before the addition of the screening compounds; (ii) Automatized capture of a low magnification brightfield image at the center of each well as a control of the general cytotoxicity of every treatment; (iii) Lysis in benzonase w/o boiling of the samples and direct loading of the samples to the SDS Page gel. c Layout of the 96 multi-well (MW) plates used in the screening, showing the disposition of the non-irradiated and UV-irradiated controls. Eighty kinase inhibitors per plate were evaluated and eight mini-western blots were run in parallel with the 12 samples from each plate row. d Results of the screening with 627 kinase inhibitors from the PKIS2 library, tested at 1 μM. The distribution of the normalized ubi-PCNA/total PCNA ratios is shown. The dotted line represents the threshold of three standard deviations that allowed the identification of 22 hits

Article Snippet: Primary antibodies used were: α-ubiquityl-PCNA (D5C7P; Cat# 13439), α-PCNA (PC-10; Cat# 2586), α-pan-Akt (Cat# 4691), α-phospho-Akt (Ser473; Cat# 9271), α-phospho-GSK3B (Ser9; Cat# 9336), α-phospho-PRAS40 (Thr246; Cat# 2997), α-RAD18 (Cat# 9040) and α-SMC-1 (Cat# 4802) from Cell Signaling Technology; α-BRCA1 (Ab-1) from Oncogene Research; α-PCNA (PC-10, Cat# sc-56) from SCBT; α-γH2AX (Cat# 05-636-1) from Millipore; α-CPD (Cat# NMDND001) from Cosmo Bio; α-Tubulin (Cat# T9026) from Sigma-Aldrich.

Techniques: Western Blot, Irradiation, Bioprocessing, Control, Lysis, SDS Page

Journal: Oncogene

Article Title: AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

doi: 10.1038/s41388-019-0724-7

Figure Lengend Snippet: AKT inhibition impairs PCNA ubiquitylation. a U2OS cells were UV irradiated (15 J/m 2 ) and treated for 12 h with the indicated inhibitors at 1 μM. The western blot shows the strong PCNA ubiquitylation inhibitory activity found in two structurally related hits: C11 (compound #: GSK1581428A) and G8 (compound #: GSK1389063A). The graph in the lower panel shows the quantification of three independent experiments. Statistical analysis was performed using analysis of variance (ANOVA) with Tukey Kramer post-test (*** p ≤ 0.001). b U2OS cells were treated as in a and western blots with specific antibodies were performed to study pAKT, total AKT, and p-GSK3β levels. α-Tubulin was used as a loading control. c U2OS cells were pre-treated for 12 h using 0.5 µM C11 and 5 µM of the structurally unrelated AKT inhibitors: MK-2206 (Merck), AZD5363 (AstraZeneca), GSK690693 (GlaxoSmithKline). After UV, all these inhibitors were used at 20 µM and C11 was used at 1 µM. The normalized ubi-PCNA/total PCNA ratios are shown below the PCNA panel. pAKT, AKT, p-GSK3β, and p-PRAS40 western blots were performed at 3 and 12 h post-treatment to confirm the AKT inhibitory activity of each compound. d U2OS cell were transfected with two concentrations of siRNAs. Forty-eight hours later, cells were UV irradiated, and after 12 h, samples were processed for quantification of PCNA ubiquitylation by western blot. A western blot for pan-AKT was performed to confirm the siRNA-mediated knockdown. The normalized ubi-PCNA/total PCNA ratios are shown below the PCNA panel

Article Snippet: Primary antibodies used were: α-ubiquityl-PCNA (D5C7P; Cat# 13439), α-PCNA (PC-10; Cat# 2586), α-pan-Akt (Cat# 4691), α-phospho-Akt (Ser473; Cat# 9271), α-phospho-GSK3B (Ser9; Cat# 9336), α-phospho-PRAS40 (Thr246; Cat# 2997), α-RAD18 (Cat# 9040) and α-SMC-1 (Cat# 4802) from Cell Signaling Technology; α-BRCA1 (Ab-1) from Oncogene Research; α-PCNA (PC-10, Cat# sc-56) from SCBT; α-γH2AX (Cat# 05-636-1) from Millipore; α-CPD (Cat# NMDND001) from Cosmo Bio; α-Tubulin (Cat# T9026) from Sigma-Aldrich.

Techniques: Inhibition, Irradiation, Western Blot, Activity Assay, Control, Transfection, Knockdown

Journal: Oncogene

Article Title: AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

doi: 10.1038/s41388-019-0724-7

Figure Lengend Snippet: AKT inhibition impairs PCNA ubiquitylation only in the context of replication stress. a U2OS cells were pre-treated overnight with the indicated kinase inhibitors: PI3K inhibitor (LY294002 50 µM), ATR inhibitor (VE-821 1 µM), ATM inhibitor (KU-55933 1 µM), DNA PKcs inhibitor (NU7026 20 µM), and AKT inhibitor (C11 1 µM). Cells were then submitted to UV irradiation (15 J/m 2 ), and 12 h in the presence of the inhibitors at the same concentrations, samples were processed by WB for the quantification of ubi/PCNA. b U2OS cells were treated in parallel and in combination of suboptimal doses of the AKT inhibitor C11 (0.1 µM) and the optimal dose of the DNA PKcs inhibitor NU7026 (20 µM). Twelve hours after UV irradiation (15 J/m 2 ), samples were processed by WB for the quantification of ubi/PCNA. c U2OS cells were treated overnight with the optimal dose of each kinase inhibitor (LY294002 50 µM, NU7026 20 µM, and C11 1 µM) followed by sample processing for WB in unperturbed conditions. d U2OS cells were transfected with 75 nM of siRNA against USP1. Forty-eight hours later, cells were treated with C11 1 µM and after 12 h, samples were processed for quantification of PCNA ubiquitylation by western blot

Article Snippet: Primary antibodies used were: α-ubiquityl-PCNA (D5C7P; Cat# 13439), α-PCNA (PC-10; Cat# 2586), α-pan-Akt (Cat# 4691), α-phospho-Akt (Ser473; Cat# 9271), α-phospho-GSK3B (Ser9; Cat# 9336), α-phospho-PRAS40 (Thr246; Cat# 2997), α-RAD18 (Cat# 9040) and α-SMC-1 (Cat# 4802) from Cell Signaling Technology; α-BRCA1 (Ab-1) from Oncogene Research; α-PCNA (PC-10, Cat# sc-56) from SCBT; α-γH2AX (Cat# 05-636-1) from Millipore; α-CPD (Cat# NMDND001) from Cosmo Bio; α-Tubulin (Cat# T9026) from Sigma-Aldrich.

Techniques: Inhibition, Irradiation, Transfection, Western Blot

Journal: Oncogene

Article Title: AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

doi: 10.1038/s41388-019-0724-7

Figure Lengend Snippet: AKT inhibition is synthetic lethal with the homologous recombination (HR) deficiency induced by BRCA1 knockdown. a Experimental layout and detailed protocol used to assess synthetic lethality (SL) induction using a co-culture method of HR+ and HR– isogenic HCT116 p21-/- cell lines, generated by lentiviral transduction of shRNAs against BRCA1. Each cell line co-expresses a different fluorescent protein: shSCR (CFP) and shBRCA1 (iRFP). Equal numbers of both isogenic cells were then plated in triplicates in 96 MW plates and combinations of increasing UV doses with AKT inhibitors were performed. Six days post-treatment, the co-cultured population was counted and categorized by the differential expression of fluorescent proteins using automated flow cytometry with an autosampler. The remaining % of each cell population was determined and the ratio of HR–/HR+ cells was calculated. The relative survival of each cell population in comparison with the untreated controls was determined to calculate SL induction by the different treatments. b Positive control to calibrate the robustness of the SL induction assay at 6 days using the PARP inhibitor Olaparib (0.1 μM), which is selectively toxic against the HR– population. c Determination of SL induction using HR+ (shSCR) and HR– (shBRCA1) isogenic HCT116 p21-/- cells in a dose-response UV irradiation curve combined with three AKT inhibitors after 6 days of treatment: C11 (0.1 µM), MK-2206 (1 µM) and AZD5363 (1 µM). Statistical analysis shown in figures b and c was performed using analysis of variance (ANOVA) (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). d Clonogenic experiments comparing HCT116 p21-/- shSCR vs. shBRCA1 cells treated with the combination of AKT inhibition (C11 0.1 µM) and UV irradiation. Duplicates of each treatment are displayed. Seven hundred fifty cells were plated in a 96 MW format and after 6 days the survival fraction was stained with crystal violet. e Control WBs confirming the efficient impairment of PCNA ubiquitylation after UV triggered by AKT inhibition for every cell line used to validate the induction of SL. In the case of mouse and hamster cells (MEF and V-C8), the detection of PCNA ubiquitylation was performed using the total PCNA antibody (PC-10) because the Ubiquityl-PCNA antibody (D5C7P) only reacts with human samples. f Determination of SL induction using HR+ and HR– cells (BRCA1 or BRCA2 deficient) at the optimal UV irradiation dose in combination with the AKT inhibitor C11 (1 µM). A pair of triple-negative breast cancer cell lines (MDA-MB 231-BRCA1wt vs MDA-MB 436-BRCA1KO), a set of wt mouse embryonic fibroblast (MEF) (shSCR vs shBRCA1), a hamster BRCA2 KO cell line with its reconstituted counterpart (V-C8 vs VC#13) and a pair of HCT116 p21-/- cells (shSCR vs shBRCA2) were used. Statistical analysis shown in panels b , c , and f was performed using analysis of variance (ANOVA) (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001)

Article Snippet: Primary antibodies used were: α-ubiquityl-PCNA (D5C7P; Cat# 13439), α-PCNA (PC-10; Cat# 2586), α-pan-Akt (Cat# 4691), α-phospho-Akt (Ser473; Cat# 9271), α-phospho-GSK3B (Ser9; Cat# 9336), α-phospho-PRAS40 (Thr246; Cat# 2997), α-RAD18 (Cat# 9040) and α-SMC-1 (Cat# 4802) from Cell Signaling Technology; α-BRCA1 (Ab-1) from Oncogene Research; α-PCNA (PC-10, Cat# sc-56) from SCBT; α-γH2AX (Cat# 05-636-1) from Millipore; α-CPD (Cat# NMDND001) from Cosmo Bio; α-Tubulin (Cat# T9026) from Sigma-Aldrich.

Techniques: Inhibition, Homologous Recombination, Knockdown, Co-Culture Assay, Generated, Transduction, Cell Culture, Quantitative Proteomics, Flow Cytometry, Comparison, Positive Control, Irradiation, Staining, Control

Journal: Oncogene

Article Title: AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

doi: 10.1038/s41388-019-0724-7

Figure Lengend Snippet: Abrogation of PCNA ubiquitylation triggers synthetic lethality in homologous recombination (HR)-deficient cells submitted to replication stress. a HCT116 p21−/− cells were transduced with four different shRNAs against RAD18 at increasing multiplicities of infection (MOI). Seventy-two hours later, cells were processed for western blot to detect RAD18. b HCT116 p21-/- cells were transduced with the most efficient RAD18 shRNAS (#1 and #4). After 72 h, cells were UV irradiated (15 J/m 2 ) and the induction of PCNA ubiquitylation was analyzed by western blot 12 h later. c Detailed protocol used to assess SL induction after RAD18 knockdown. Cells were plated in 6 MW plates and transduced with lentiviral shRNAs #1 or #4. Forty-eight hours later, cells were re-plated into a 96 MW format. Twenty-four hours later, cells were UV irradiated. Six days post UV irradiation, the determination of SL induction using HR+ and HR– cells was performed by calculating the relative survival of the cells transduced with the shRNA against RAD18 in comparison with the non-transduced cells. Such differential analysis was possible by gating the transduced population due to the concomitant expression of GFP with the shRNAs. d HR+ and HR- cells were transduced with shRNA #1 using the protocol detailed in ( c ). The relative survival of the transduced population was calculated using the non-irradiated population as control. A dose-response UV curve was performed, and samples were processed using eight experimental replicates. e PCNA wt and PCNA K164R mouse embryonic fibroblasts (MEFs) were UV irradiated (40 J/m 2 ). Twelve hours after UV irradiation, samples were processed for WB and ubi-PCNA induction was assessed using a monoclonal antibody that detects mouse PCNA. f PCNA wt and PCNA K164R MEFs were transduced with shRNAs against murine BRCA1. Each set of cells were UV irradiated following a dose-response curve with or without treatment with the AKT inhibitor C11. After 6 days, the relative survival of each cell population was determined using automated flow cytometry. g HR+ and HR– cell pairs were treated with cisplatin and after 1 h the culture media was replaced. The relative survival of the different HR– vs HR+ pairs was assessed 6 days later. h PCNA wt and PCNA K164R MEFs were treated with cisplatin and after 1 h the culture media was replaced. The relative survival of the different HR– vs HR+ pairs was assessed 6 days later. Statistical analysis shown in panels d , f , g , and h was performed using analysis of variance (ANOVA) (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001)

Article Snippet: Primary antibodies used were: α-ubiquityl-PCNA (D5C7P; Cat# 13439), α-PCNA (PC-10; Cat# 2586), α-pan-Akt (Cat# 4691), α-phospho-Akt (Ser473; Cat# 9271), α-phospho-GSK3B (Ser9; Cat# 9336), α-phospho-PRAS40 (Thr246; Cat# 2997), α-RAD18 (Cat# 9040) and α-SMC-1 (Cat# 4802) from Cell Signaling Technology; α-BRCA1 (Ab-1) from Oncogene Research; α-PCNA (PC-10, Cat# sc-56) from SCBT; α-γH2AX (Cat# 05-636-1) from Millipore; α-CPD (Cat# NMDND001) from Cosmo Bio; α-Tubulin (Cat# T9026) from Sigma-Aldrich.

Techniques: Homologous Recombination, Transduction, Infection, Western Blot, Irradiation, Knockdown, shRNA, Comparison, Expressing, Control, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Engineering of α-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration

doi: 10.1038/s41419-020-03187-1

Figure Lengend Snippet: a Schematic overview of the donor plasmid, Cas9/sgRNA expression plasmid, and targeting strategy for α-PD-1 integration into GAPDH 3′-UTR. Positions of the PCR primers (black arrows) used for detection of integrated DNA fragments are indicated. Fine gray lines on donor plasmids indicate sections homologous to the GAPDH locus. Lightning shape, sgRNA target sequence, HR, homologous recombination-based strategy, HMEJ, homology-mediated end joining-based strategy, LHR/RHR, left/right arm of homology recombination, F1/R2, outer forward/reverse primer, F2/R1, inner forward/reverse primer. b The mismatch-sensitive endonuclease T7E1 assay results showed the different efficiencies of Cas9/sgRNA-1, 2, and 3 for targeting human HEK293T genome. HEK293T cells were transfected with Cas9/sgRNA-1, 2 or 3 expression plasmid, without donor plasmid. Genomic DNA was extracted for T7E1 assay at day 4 post transfection. c FACS analysis of HEK293T cells showed the knock-in efficiencies of the α-PD-1 mAb in HEK293T cells. IDLV with HR-donor alone, IDLV expressing Cas9/sgRNA alone, or the two IDLVs together, were allowed to infect HEK293T cells. Control without IDLV infection is shown on the top. d CD90 + cells were sorted for genomic PCR analysis. Two sets of primers specific for the 5′ or 3′ integration junctions were used. Primer pair F1/R1 and F2/R2 amplified the 5′-junction (1435 bp) and the 3′-junction (1008 bp) of the transgene integration respectively. Primers F1/R2 amplified two DNA fragments that represent the wild type (2176 bp) and modified gene (4929 bp), respectively. e Relative knock-in efficiencies of HR and HMEJ-based strategies in HEK293T cells. Cells were infected with IDLV carried HR-donor or HMEJ-donor along with IDLV expressing Cas9/sgRNA at different MOIs. CD90 expression was analyzed by FACS 5 days post infection. Data are representative of three independent experiments (means ± SEM), ** P < 0.01, ns, no significant difference; two-tailed Student’s t test ( e ) was used.

Article Snippet: Primary antibodies used were as follows: mouse anti-human CD90 antibody (Clone 7E1B11, ab181469, Abcam) and rabbit anti-human Syndecan-1 antibody (Clone SP152, ab130405, Abcam).

Techniques: Plasmid Preparation, Expressing, Sequencing, Homologous Recombination, Transfection, Knock-In, Control, Infection, Amplification, Modification, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Engineering of α-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration

doi: 10.1038/s41419-020-03187-1

Figure Lengend Snippet: a Comparison of transduction rates between BaEVTR and VSVG pseudotyped IDLVs. An incubation of the freshly pre-stimulated B cells with BaEVTR or VSVG pseudotyped GFP-encoding IDLVs (MOI of 10, based on titering via flow cytometry for GFP expression) was conducted for 48 h at 37 °C, followed by FACS analysis for detection of GFP + cells. Pre-stimulated B cells without transduction were used as the control. b A wave of transgene expression was observed at human primary B cells transduced with BaEVTR pseudotyped IDLV. GFP expression was estimated by FACS at 24 h, day 7 or day 14 post transduction. c Detection of BaEVTR pseudotyped ICLVs and IDLVs integration at 24 h or day 14 post transduction with a modified Alu-LTR nested–PCR protocol. Results are presented as mean ± SEM, n = 3. d Schematic representation of the human primary B cells engineering protocol by infection of dual-IDLVs. e Representative flow cytometric analysis for integrated CD90 expression 5 days post infection as indicated in d . Pre-stimulated B cells with only the donor IDLV were used as the control. f The relative knock-in efficiency in human primary B cells transduced with dual-IDLVs are shown. Data are from three independent experiments (means ± SEM), *** P < 0.001; two-tailed Student’s t test ( f ) was used.

Article Snippet: Primary antibodies used were as follows: mouse anti-human CD90 antibody (Clone 7E1B11, ab181469, Abcam) and rabbit anti-human Syndecan-1 antibody (Clone SP152, ab130405, Abcam).

Techniques: Comparison, Transduction, Incubation, Flow Cytometry, Expressing, Control, Modification, Nested PCR, Infection, Knock-In, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Engineering of α-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration

doi: 10.1038/s41419-020-03187-1

Figure Lengend Snippet: a Schematic representation of sorting and expansion strategy of engineered CD90-expressing B cells. b Following FACS sorting at day 0 indicated in a , engineered CD90-expressing B cells were co-cultured with the irradiated 293T, 293T-CD40L, or 293T-CD40L-sBAFF feeder cells. The feeder cells were renewed every 4 days and the numbers of B cells were counted to indicate expansion patterns. c As performed in a , culture supernatants of gene-edited B cells co-cultured with feeder cells were collected at various time points, followed by ELISA for detecting α-PD-1 mAb concentration. Data are representative of three independent experiments. d PD-1 blockade-mediated T-cell stimulation assay was performed by SEB stimulation of PBMCs. 1 × 10 5 PBMCs were stimulated with serial dilutions of SEB in the presence of nivolumab, culture supernatant of gene-edited B cells or untransduced B cells as a control. Supernatants were collected 3 days later and measured for IL-2 levels by ELISA. Nivolumab was used as a positive control. Representative data from three healthy donors are shown. e To conduct a T-cell proliferation assay mediated by PD-1 blockade, PBMCs from healthy donors were stimulated with anti-CD3 antibody and cultured in the presence of anti-CD28, nivolumab, culture supernatant of gene-edited B cells or untransduced B cells as a control for 3 days. The CFSE labeled CD4 + T cells were detected via flow cytometry. f Genome alignment tracks of the normalized ATAC-seq data showed the open chromatin for GAPDH locus in CD90 + engineered B cells cultured at day 28 (red). Pre-sitmulated B cells with only the donor IDLV were used as the control (blue). The results in panels c , d , and e are presented as mean ± SEM, n = 3. ** P < 0.01, *** P < 0.001, ns, no significant difference; one-way ANOVA with Tukey’s post hoc tests ( c – e ) were used.

Article Snippet: Primary antibodies used were as follows: mouse anti-human CD90 antibody (Clone 7E1B11, ab181469, Abcam) and rabbit anti-human Syndecan-1 antibody (Clone SP152, ab130405, Abcam).

Techniques: Expressing, Cell Culture, Irradiation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Stimulation, Control, Positive Control, Proliferation Assay, Labeling, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Engineering of α-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration

doi: 10.1038/s41419-020-03187-1

Figure Lengend Snippet: a The strategy of investigating long-term efficacy of LLPCs combined with trametinib (Tra) and dabrafenib (Dab) treatment against human A375 melanoma. Mice treated only with vehicle (PBS, pH 7.0) were used as the control. Trametinib and dabrafenib were administrated every 2 days for 10 days. Nivolumab was given three times every week. b Tumor growth curves. Treatments began at day 10. Removing of inhibitors is marked by the black arrow. Representative graph of two repetitions of this experiment is shown. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01; one-way ANOVA with Tukey’s post hoc test ( b ) was used. c The tumor infiltrating hCD3 + and hCD8 + lymphocytes. The melanoma biopsies were formalin fixed and processed for immunohistochemistry analysis at day 42. Anti-human CD3 antibody and anti-human CD8 antibody were used for primary staining (scale bar, 100 µm). d Representative flow cytometric analysis of LLPCs proportion in the spleen and representative immunofluorescence images indicating the engineered LLPCs population in the bone marrow. Human CD138 is shown in red, human CD90 in green, and DAPI-stained nuclei in blue. Scale bar, 10 μm.

Article Snippet: Primary antibodies used were as follows: mouse anti-human CD90 antibody (Clone 7E1B11, ab181469, Abcam) and rabbit anti-human Syndecan-1 antibody (Clone SP152, ab130405, Abcam).

Techniques: Control, Immunohistochemistry, Staining, Immunofluorescence

Journal: Cancer Medicine

Article Title: EGFR confers radioresistance in human oropharyngeal carcinoma by activating endoplasmic reticulum stress signaling PERK‐eIF2α‐GRP94 and IRE1α‐XBP1‐GRP78

doi: 10.1002/cam4.1862

Figure Lengend Snippet: EGF‐EGFR activates ERS signaling. A, Total and phosphorylated PERK, eIF2α and IRE1α as well as GRP78, GRP94, and spliced XBP‐1 in control and EGFR siRNA transfected FaDuR, Detroit562R, FaDuP, and Detroit562P cells, 12 h after 5 Gy of irradiation. Also shown is total ATF6 (90 kDa) and cleaved ATF6 (50 kDa) in FaDuR, Detroit562R, FaDuP, and Detroit562P cells, 12 h after 5 Gy of irradiation. B, GRP94 and GRP78 protein levels in control and PERK and IRE1α siRNA transfected FaDuR, Detroit562R, FaDuP, and Detroit562P cells. C, Quantitative real‐time PCR analysis of PERK and IRE1α mRNA in control and EGFR siRNA transfected FaDuR, Detroit562R, FaDuP, and Detroit562P cells 1 h after 5 Gy of irradiation. As shown, EGFR silencing inhibited irradiation‐induced PERK and IRE1α mRNA expression. Note: * P < 0.05 and # P < 0.01 compared to the irradiated group. D, Total and phosphorylated PERK, eIF2α and IRE1α as well as GRP78, GRP94, and spliced XBP‐1 in control and EGFR siRNA transfected FaDuR, Detroit562R, FaDuP, and Detroit562P cells pre‐treated with 10 ng/mL EGF followed by 5 Gy of irradiation. Also shown is total and cleaved ATF6 in FaDuR, Detroit562R, FaDuP, and Detroit562P cells treated with 10 ng/mL EGF followed by 5 Gy of irradiation. Note: For A, B, and D, the protein bands were quantified using ImageJ software and normalized to β‐actin. Fold changes are shown compared to the negative control without radiation. N/A = not applicable

Article Snippet: The membranes were blocked in 5% non‐fat milk in 1× TBST and then incubated with primary antibodies against EGFR (1:500; Santa Cruz, USA), PERK, IRE1α, ATF 6, (1:1000; Abcam), phospho‐eIF2α, GRP78, GRP94, PDI, ERO1‐Lα, CHOP, phospho‐ATM, DNA‐PK, LC3B, Atg3, cleaved caspase 3, cleaved PARP, and β‐actin (1:1000; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight.

Techniques: Control, Transfection, Irradiation, Real-time Polymerase Chain Reaction, Expressing, Software, Negative Control

Journal: Cancer Medicine

Article Title: EGFR confers radioresistance in human oropharyngeal carcinoma by activating endoplasmic reticulum stress signaling PERK‐eIF2α‐GRP94 and IRE1α‐XBP1‐GRP78

doi: 10.1002/cam4.1862

Figure Lengend Snippet: ERS pathway activation regulates EGF‐EGFR‐mediated cell proliferation and radiosensitivity of OSCC cells. A, Total and phosphorylated ERK and AKT at 0‐24 h in FaDuR, Detroit562R, FaDuP, and Detroit562P after 5 Gy of irradiation. B, EGR1 and Bcl‐2 levels in control and EGF (10 ng/mL, 2 h) treatment FaDuR, Detroit562R, FaDuP, and Detroit562P 12 h after 5 Gy of irradiation pre‐treated with or without (IRE1 inhibitor) 4μ8C (10 μmol/L), (PERK inhibitor) GSK2606414 (1 μmol/L), (ERK inhibitor) U0126 (20 μmol/L), and (AKT inhibitor) SH‐5 (10 μmol/L). C, CCK‐8 cell proliferation assay of control and EGF (10 ng/mL, 2 h) treatment FaDuR, Detroit562R, FaDuP, and Detroit562P 48 h after 5 Gy of irradiation pre‐treated with or without (IRE1 inhibitor) 4μ8C (10 μmol/L), (PERK inhibitor) GSK2606414 (1 μmol/L), (ERK inhibitor) U0126 (20 μmol/L), and (AKT inhibitor) SH‐5 (10 μmol/L). Note: * denotes P < 0.05, and # denotes P < 0.01 compared to the irradiated group. D, Total and phosphorylated ERK and AKT in control and EGF (10 ng/mL) treatment with or without PERK and IRE1α siRNA transfected FaDuR, Detroit562R, FaDuP, and Detroit562P cells 12 h after 5 Gy of irradiation

Article Snippet: The membranes were blocked in 5% non‐fat milk in 1× TBST and then incubated with primary antibodies against EGFR (1:500; Santa Cruz, USA), PERK, IRE1α, ATF 6, (1:1000; Abcam), phospho‐eIF2α, GRP78, GRP94, PDI, ERO1‐Lα, CHOP, phospho‐ATM, DNA‐PK, LC3B, Atg3, cleaved caspase 3, cleaved PARP, and β‐actin (1:1000; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight.

Techniques: Activation Assay, Irradiation, Control, CCK-8 Assay, Proliferation Assay, Transfection

Journal: Cancer Medicine

Article Title: EGFR confers radioresistance in human oropharyngeal carcinoma by activating endoplasmic reticulum stress signaling PERK‐eIF2α‐GRP94 and IRE1α‐XBP1‐GRP78

doi: 10.1002/cam4.1862

Figure Lengend Snippet: EGFR and PERK expression in human OSCC tissues. Representative images of immunohistochemically stained OSCC patient tissues showing A, high and B, low EGFR and PERK expression with 400× magnification. C, Kaplan‐Meier survival curve analysis showing correlation between the OS and EGFR and PERK expression in 80 OSCC patients

Article Snippet: The membranes were blocked in 5% non‐fat milk in 1× TBST and then incubated with primary antibodies against EGFR (1:500; Santa Cruz, USA), PERK, IRE1α, ATF 6, (1:1000; Abcam), phospho‐eIF2α, GRP78, GRP94, PDI, ERO1‐Lα, CHOP, phospho‐ATM, DNA‐PK, LC3B, Atg3, cleaved caspase 3, cleaved PARP, and β‐actin (1:1000; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight.

Techniques: Expressing, Staining

Journal: PloS one

Article Title: Modulation of radiation injury response in retinal endothelial cells by quinic acid derivative KZ-41 involves p38 MAPK.

doi: 10.1371/journal.pone.0100210

Figure Lengend Snippet: Figure 4. Induction of p38MAPK phosphorylation in irradiated RECs. A) RECs receiving 30 Gy irradiation in a single fraction show increases in phosphorylation of p38MAPK (T180/Y182) that reach a plateau between 4–8 hours compared to control cells at same time-points (*P,0.05). B) Irradiated RECs with or without treatment of KZ-41 (10 mM) were harvested and analyzed for phospho-p38MAPK at 4 hours. KZ-41 treated RECs showed significant reductions in total levels of phosphorylated p38MAPK (T180/Y182), as compared to IR-RECs (**P,0.05). doi:10.1371/journal.pone.0100210.g004

Article Snippet: Phosphorylated (Thr180/Tyr182) p38MAPK primary antibodies were purchased from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Irradiation, Control

Journal: PloS one

Article Title: Modulation of radiation injury response in retinal endothelial cells by quinic acid derivative KZ-41 involves p38 MAPK.

doi: 10.1371/journal.pone.0100210

Figure Lengend Snippet: Figure 6. Radiation-induced paxillin-dependent proliferative capacity/phenotype. A) Irradiation-induced REC proliferation was measured after 24 hours using the WST-1 proliferation assay. REC proliferation was enhanced by irradiation and was significantly reduced with treatment of KZ- 41 (10 mM) (*P,0.05). B) Paxillin phosphorylation (Y118) was measured 24 hours after irradiation using immunoblotting and showed enhanced levels. Both KZ-41 (10 mM) and p38MAPK inhibitor SB202190 (10 mM) significantly reduced levels of paxillin phosphorylation (*P,0.01, #P,0.05). doi:10.1371/journal.pone.0100210.g006

Article Snippet: Phosphorylated (Thr180/Tyr182) p38MAPK primary antibodies were purchased from R&D Systems (Minneapolis, MN).

Techniques: Irradiation, Proliferation Assay, Phospho-proteomics, Western Blot

Journal: PloS one

Article Title: Modulation of radiation injury response in retinal endothelial cells by quinic acid derivative KZ-41 involves p38 MAPK.

doi: 10.1371/journal.pone.0100210

Figure Lengend Snippet: Figure 9. Model of KZ-41 radioprotective mechanism-of-action. Gamma-(c) radiation-induced DNA double strand breaks (DSBs) trigger phosphorylation of p38MAPK which in turn results in p53 accumulation enhances ICAM-1 surface levels and incites a proliferative/migratory phenotype through paxillin phosphorylation. KZ-41 reduces phospho-p38MAPK and effectively uncouples p38 MAPK signaling to reduce REC inflammation and halt aberrant cellular motility. Therefore, KZ-41 is able to protect RECs against acute radiation injury and the resultant dysfunction of the retinal vasculature. doi:10.1371/journal.pone.0100210.g009

Article Snippet: Phosphorylated (Thr180/Tyr182) p38MAPK primary antibodies were purchased from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics

Journal: Journal of Cancer

Article Title: Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma

doi: 10.7150/jca.36164

Figure Lengend Snippet: FL BARD1 expression correlates with expression of genes involved in the cell cycle and DNA repair . Gene Ontology results for genes that significantly correlated with FL BARD1 expression (RNAseq) in 161 neuroblastoma tumors. P-value is reported as -Log10 (P) on X-axis.

Article Snippet: Primary antibodies used are: anti-human BARD1 (cod-A300-263A, Bethyl, 1:1000), γH2AX (phosphoSer139) (cod-H5912, Sigma Aldrich, 1:1000), phosphor-p53(Ser-15) (cod-9284 Cell Signaling, 1:500), p53 (sc-6243, Santa Cruz, 1:500), Cyclin B (sc-752 Santa Cruz, 1:500), CDK1 (sc-54, Santa Cruz, 1:1000); phospho-H3 (06-570 Millipore).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma

doi: 10.7150/jca.36164

Figure Lengend Snippet: FL BARD1 functions in DNA damage response. SKNSH and SHSY5Y cell lines were silenced for FL BARD 1 expression upon transfection with lentiviral plasmids (shBARD1#A, shBARD1#B). Unsilenced control cells were transfected with plasmid shCTR. The efficiency of short harpin silencing was verified by western blotting, using an antibody against FL BARD1 isoform. The molecular weight of FL BARD1 isoform is reported. The higher band in the blot is an aspecific staining. β-Actin levels were used as loading control (A). The detection of Υ -H2AX protein was verified in nuclear extract of silenced (shBARD1) and unsilenced control (shCTR) cells, by western blotting. Antibody against histone H3 was used as loading control (B). SKNSH shBARD1 and shCTR cells (C) and SHSY5Y shBARD1 and shCTR cells (D) were treated with 5 Gy IR. The expression of Υ- H2AX was measured by western blotting in a time-course (30 min, 1h, 3h, 6h, 12h, 24h, 36h, 48h) after IR. The integral optical density (IOD) of Υ- H2AX protein bands were measured and normalized respect to loading control protein band H3. The arrows indicate the higher increment of Υ- H2AX in each cell line. The experiments were repeated twice.

Article Snippet: Primary antibodies used are: anti-human BARD1 (cod-A300-263A, Bethyl, 1:1000), γH2AX (phosphoSer139) (cod-H5912, Sigma Aldrich, 1:1000), phosphor-p53(Ser-15) (cod-9284 Cell Signaling, 1:500), p53 (sc-6243, Santa Cruz, 1:500), Cyclin B (sc-752 Santa Cruz, 1:500), CDK1 (sc-54, Santa Cruz, 1:1000); phospho-H3 (06-570 Millipore).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Molecular Weight, Staining

Journal: Journal of Cancer

Article Title: Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma

doi: 10.7150/jca.36164

Figure Lengend Snippet: FL BARD1 functions in G2-M cell cycle phase. Cyclin B and CDK1 protein levels were verified in cytosol extracts and phospho-H3 and H3 protein levels were verified in nuclei extracts by western blotting in SKNSH (24 hours post-IR) and in SHSY5Y (36 hours post-IR) cells (A). Cell cycle distribution phases were reported as mean percentages between two experiments, in SKNSH shBARD1 and shCTR IR cells and non-irradiated cells (V) (B) and in SHSY5Y shBARD1 and shCTR IR cells and non-irradiated cells (V) (C).

Article Snippet: Primary antibodies used are: anti-human BARD1 (cod-A300-263A, Bethyl, 1:1000), γH2AX (phosphoSer139) (cod-H5912, Sigma Aldrich, 1:1000), phosphor-p53(Ser-15) (cod-9284 Cell Signaling, 1:500), p53 (sc-6243, Santa Cruz, 1:500), Cyclin B (sc-752 Santa Cruz, 1:500), CDK1 (sc-54, Santa Cruz, 1:1000); phospho-H3 (06-570 Millipore).

Techniques: Western Blot, Irradiation

Journal: Journal of Cancer

Article Title: Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma

doi: 10.7150/jca.36164

Figure Lengend Snippet: FL BARD1 functions in regulating apoptosis. Phospho-p53 and p53 and β-Actin protein levels were verified by western blotting in shBARD1 and shCTR cells, in SKNSH (24 hours post-IR) and SHSY5Y (36 hours post-IR) cell lines (A). Caspase-3 activity was evaluated in SKNSH shBARD1 and shCTR IR and V cells (B) and in SHSY5Y shBARD1 and shCTR IR and V cells (C). The asterisk is indicative of p-value ≤ 0.05. The experiments were repeated twice.

Article Snippet: Primary antibodies used are: anti-human BARD1 (cod-A300-263A, Bethyl, 1:1000), γH2AX (phosphoSer139) (cod-H5912, Sigma Aldrich, 1:1000), phosphor-p53(Ser-15) (cod-9284 Cell Signaling, 1:500), p53 (sc-6243, Santa Cruz, 1:500), Cyclin B (sc-752 Santa Cruz, 1:500), CDK1 (sc-54, Santa Cruz, 1:1000); phospho-H3 (06-570 Millipore).

Techniques: Western Blot, Activity Assay

Journal: Journal of Cancer

Article Title: Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma

doi: 10.7150/jca.36164

Figure Lengend Snippet: FL BARD1 depletion promotes cells proliferation and cells growth. Cell proliferation assay was performed after seven days from IR (D7). SKNSH shBARD1 (V and IR) and shCTR (V and IR) cells viability were evaluated in the following 8 (8D), 9 (9D) and 10 days (10D) from IR and normalized respect to D7 (A). Soft agar assay was performed for the same cells and colonies number for each experimental point is reported on Y-axis (B). SHSY5Y shBARD1 (V and IR) and shCTR (V and IR) cells viability were evaluated in the following 8 (8D), 9 (9D) and 10 days (10D) from IR and normalized respect to D7 as shown in (C). Soft agar assay was performed for the same cells and colonies number for each experimental point in reported on Y-axis (D). The asterisks show the increments of colonies number with P<0.05 in shBARD1 V cells compared to shCTR V cells and in shBARD1 IR cells compared to shCTR IR cells (B, D). Cell viability assays were repeated twice, colony formation assays were repeated three times.

Article Snippet: Primary antibodies used are: anti-human BARD1 (cod-A300-263A, Bethyl, 1:1000), γH2AX (phosphoSer139) (cod-H5912, Sigma Aldrich, 1:1000), phosphor-p53(Ser-15) (cod-9284 Cell Signaling, 1:500), p53 (sc-6243, Santa Cruz, 1:500), Cyclin B (sc-752 Santa Cruz, 1:500), CDK1 (sc-54, Santa Cruz, 1:1000); phospho-H3 (06-570 Millipore).

Techniques: Proliferation Assay, Soft Agar Assay

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Allograft airway fibrosis in the pulmonary milieu: a disorder of tissue remodeling.

doi: 10.1111/j.1600-6143.2007.02106.x

Figure Lengend Snippet: Figure 1: Fibrotic obliteration of allograft airways in the lung. (A) Dynamic changes of cell population, matrix metalloproteinases (MMPs), lumenal obliteration and stages of fibrosis in allografts are summarized. Interventional experiments using a broad-spectrum MMP inhibitor, SC080, were planned to target early inflammation, developing fibrosis and established fibrosis. (B) In isografts, the epithelial lining was lost at day 7 and recovered by day 21. The airway was not obliterated by fibrotic tissue. In allografts, the epithelium was lost, and the airway was completely obliterated by fibrosis at day 21. (C) Immunohistochemistry for CD3+ T cells and CD68+ macrophages (brown staining). Large numbers of T cells and macrophages in allograft lumen at days 7 decreased over time. (D) Morphometric quantification of obliterative fibrosis demonstrated significant differences between isografts and allografts after day 14 (∗p < 0.05; data are expressed as mean ± SEM; n = 4). (E) Fibrosis established in allografts persisted until day 98. Scale bars = 50 lm. Original magnification of insets was 40×.

Article Snippet: After deparaffinization and antigen retrieval in boiling 0.01 M citrate buffer (pH 6) for 20 minutes for paraffin sections, all sections were blocked and incubated overnight at 4◦C with primary antibodies including mouse anti-rat CD68 (1:100, Serotec, Raleigh, NC), rabbit anti-human CD3 (1:100, DAKO Canada, Inc., Mississauga, Canada), goat anti-procollagen a1(I) (1:50, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit anti-MMP-2, -3, -9 and -13 (1:50, Biomol, Plymouth Meeting, PA), and rabbit anti-MMP-14 (prediluted, Abcam, Cambridge, MA).

Techniques: Immunohistochemistry, Staining

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Allograft airway fibrosis in the pulmonary milieu: a disorder of tissue remodeling.

doi: 10.1111/j.1600-6143.2007.02106.x

Figure Lengend Snippet: Figure 5: SC080 treatment from day 0 to day 28 inhibits lumenal leukocyte infiltration, but not T-cell activation in allografts. (A) SC080 treatment prevented cell infiltration in graft lumen. Original magnification of insets was 40×. (B) Quantification of lumenal cell infiltration demonstrated significantly less numbers of cells in the SC080-treated allografts at day 7 (∗p < 0.001). Data are expressed as mean ± SEM. (C) At day 7, in the sub-epithelial area SC080 treatment did not reduce total cell infiltration (hematoxylin and eosin stain), CD68+ macrophages, or CD3+ T cells. Arrow heads indicate epithelium or the location that should contain epithelium. (D) Flow cytometry analysis at day 10 using whole graft extract. (a) SC080 treatment did not reduce infiltration of total T-cell number (CD3+) and T-cell subsets (CD4+ and CD8+ T cells) in allografts. The number of cells was significantly less in isografts. (b) SC080 treatment did not reduce the CD8/CD4 ratio in allografts, suggesting an active cellular immune response in allografts either with or without SC080 treatment. (E) (i) T-cell proliferation assay. Lymphocytes (1 × 105) from draining lymph nodes of nontransplanted Lewis rats stimulated with plate-bound anti-rat CD3 and anti-rat CD28 with doses of indicated drugs. (ii) Mixed lymphocyte reaction by transplant recipients. Cells (1 × 105) from the spleen of SC080-treated versus nontreated transplanted (Tx, postoperative day 10) animals were used as responders against indicated numbers of irradiated allo-splenocytes (Brown-Norway) and cultured for 4 days. (F) Real-time RT-PCR analysis for tracheal allografts at day 7 with or without SC080 treatment. Gene expression of interferon (IFN)-c was significantly increased by SC080 whereas IL-1b was significantly decreased by SC080. (∗represents significant differences compared to other groups, p < 0.05, Tukey test; Scale bars = 50 lm.)

Article Snippet: After deparaffinization and antigen retrieval in boiling 0.01 M citrate buffer (pH 6) for 20 minutes for paraffin sections, all sections were blocked and incubated overnight at 4◦C with primary antibodies including mouse anti-rat CD68 (1:100, Serotec, Raleigh, NC), rabbit anti-human CD3 (1:100, DAKO Canada, Inc., Mississauga, Canada), goat anti-procollagen a1(I) (1:50, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit anti-MMP-2, -3, -9 and -13 (1:50, Biomol, Plymouth Meeting, PA), and rabbit anti-MMP-14 (prediluted, Abcam, Cambridge, MA).

Techniques: Activation Assay, H&E Stain, Flow Cytometry, Proliferation Assay, Irradiation, Cell Culture, Quantitative RT-PCR, Gene Expression

Journal: JACC: Basic to Translational Science

Article Title: miR-29b Mediates the Chronic Inflammatory Response in Radiotherapy-Induced Vascular Disease

doi: 10.1016/j.jacbts.2018.10.006

Figure Lengend Snippet: Irradiation Affects miR-29b, miR-146b, and Target Gene Expression In Vitro; Pathological Changes Are Partly Corrigible With miRNA Mimics (A) In human carotid artery smooth muscle cells (HCtASMCs), 2 × 2 Gy of irradiation resulted in significantly reduced miR-29b expression; miR-146b expression was not affected. ∗p < 0.05. (B) The opposite was observed with human carotid artery endothelial cells (HCtAECs). ∗p < 0.05. (C) miR-29b target gene expression in HCtASMCs after irradiation. n = 6 in each group. Mean ± SEM. *p < 0.05, **p < 0.01 in Student’s t -test. (D) Western blot analysis showed an increase in PTX3 protein expression after irradiation. (E) Treatment with miR-29b mimics resulted in undetectable soluble collagen in supernatant from radiated HCtASMCs but not from control HCtASMCs. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (F) miR-29b mimic treatment reduced PTX3 expression in HCtASMCs. n = 6 in each group. Mean ± SEM. *p < 0.05, ∗∗∗∗p < 0.0001 in 1-way analysis of variance. DPP4 gene expression remained unchanged but was reduced on the protein level as shown with (G) Western blotting of HCtASMCs. Other abbreviations as in Figure 1 .

Article Snippet: Primary, proliferating human carotid artery smooth muscle cells (HCtASMCs) and human carotid artery endothelial cells (HCtAECs) (Cell Applications, San Diego, California) were cultured up to passages 5 through 8 in subtype-specific growth medium (Cell Applications).

Techniques: Irradiation, Targeted Gene Expression, In Vitro, Expressing, Western Blot, Control, Gene Expression