Journal: Scientific Reports

Article Title: Upregulated long noncoding RNA LOC105375913 induces tubulointerstitial fibrosis in focal segmental glomerulosclerosis

doi: 10.1038/s41598-018-36902-2

Figure Lengend Snippet: Role of p38 MAPK in the expression of LOC105375913 in tubular cells. ( a ) RT-PCR analysis of LOC105375913 in HK-2 cells treated with C3a, SB203580, PD098059 and MK2206 (n = 5); ( b ) RNA pull-down and RT-PCR analysis of the binding between LOC105375913 and miR-27b in HK-2 cells treated with C3a and SB203580 (n = 5); ( c ) Western blot analysis of snail, FN and Col I in HK-2 cells treated with C3a and SB203580 (n = 3); ( d,e ) Western blot analysis of p-p38 in HK-2 cells treated with C3a or plenti-CMV-LOC105375913 plasmid (n = 3); ( f ) RT-PCR analysis of LOC105375913 in HK-2 cells treated with U46619 (n = 5); ( g ) RNA pull-down and RT-PCR analysis of the binding between LOC105375913 and miR-27b in HK-2 cells treated with U46619 (n = 5); ( h ) Western blot analysis of snail, FN and Col I in HK-2 cells treated with U46619 (n = 3). For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for ( a , b and g ) and a two-tailed Student’s t-test was used for ( f ). * P < 0.05 compared with control. # P < 0.05 compared with C3a-treated cells.

Article Snippet: For intervention studies, 1 μM of C3aR antagonist SB290157 (sc-222291, Santa Cruz), 100 μg/ml of eculizumab (Soliris, Alexion Pharmaceuticals), 10 μM of p38 MAPK inhibitor SB203580 (sc-3533, Santa Cruz), 50 μM of ERK inhibitor PD98059 (sc-3532, Santa Cruz), 10 μM of Akt inhibitor MK2206 (sc-364537, Santa Cruz) or 1 μM of U-46619 (sc-201242, Santa Cruz) was given 30 min before treatments.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Western Blot, Plasmid Preparation, Two Tailed Test, Control

Journal: Scientific Reports

Article Title: Upregulated long noncoding RNA LOC105375913 induces tubulointerstitial fibrosis in focal segmental glomerulosclerosis

doi: 10.1038/s41598-018-36902-2

Figure Lengend Snippet: Role of XBP-1s in the expression of LOC105375913 in tubular cells. ( a ) RT-PCR analysis of LOC105375913 in HK-2 cells treated with C3a, XBP-1s siRNA, C/EBPβ siRNA, Elk-1 siRNA, ERα siRNA and GR siRNA (n = 3); ( b ) Level of XBP-1s and p-XBP-1s protein in HK-2 cells treated with C3a and SB203580 (n = 3); ( c ) ChIP analysis of the binding between XBP-1s and LOC105375913 promoter in HK-2 cells treated with C3a (n = 3); ( d ) Schematic of the constructed LOC105375913 promoter-luciferase reporter plasmids; ( e ) Normalized luciferase activity of reporter constructs in HK-2 cells cotransfected with XBP-1s plasmid (n = 5); ( f ) Level of LOC105375913 in HK-2 cells transfected with XBP-1s plasmid (n = 5); ( g ) RNA pull-down and RT-PCR analysis of the binding between LOC105375913 and miR-27b in HK-2 cells transfected with XBP-1s plasmid (n = 5); ( h ) Western blot analysis of snail, FN and Col I in HK-2 cells transfected with XBP-1s plasmid (n = 3); ( i ) RNA pull-down and RT-PCR analysis of the binding between LOC105375913 and miR-27b in HK-2 cells treated with C3a and XBP-1s siRNA (n = 5); ( j ) Western blot analysis of snail, FN and Col I in HK-2 cells treated with C3a and XBP-1s siRNA (n = 3). For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for ( a , e , g and i ), and a two-tailed Student’s t-test was used for ( f ). * P < 0.05 compared with control; # P < 0.05 compared with C3a-treated cells.

Article Snippet: For intervention studies, 1 μM of C3aR antagonist SB290157 (sc-222291, Santa Cruz), 100 μg/ml of eculizumab (Soliris, Alexion Pharmaceuticals), 10 μM of p38 MAPK inhibitor SB203580 (sc-3533, Santa Cruz), 50 μM of ERK inhibitor PD98059 (sc-3532, Santa Cruz), 10 μM of Akt inhibitor MK2206 (sc-364537, Santa Cruz) or 1 μM of U-46619 (sc-201242, Santa Cruz) was given 30 min before treatments.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Construct, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Western Blot, Two Tailed Test, Control

Journal: Arthritis Research & Therapy

Article Title: Tumor necrosis factor alpha and epidermal growth factor act additively to inhibit matrix gene expression by chondrocyte

doi: 10.1186/ar1464

Figure Lengend Snippet: Changes in cell morphology induced by the combination of tumor necrosis factor alpha (TNF-α) + epidermal growth factor (EGF) are partially reduced by inhibition of MEK1/2. (a) Confluent monolayers of chondrocytes were pretreated with U0124 (10 μM) or U0126 (10 μM) for 15 min followed by 24-hour treatment with TNF-α (30 ng/ml), EGF (10 ng/ml) or TNF-α + EGF. Digital images of live cultures were captured at 20 × objective magnification. Bar = 100 μm. Images are representative of two independent experiments. (b) An elongated cell is defined as having a predominant axis with a length exceeding three times the maximum width of the cell. The total number of elongated cells per field (1.376 mm 2 ) were counted, averaged for three independent experiments ( n = 3), and analyzed by analysis of variance. a Significant difference from respective control ( P < 0.05), b significant difference from respective control ( P < 0.001), c significant difference from U0124 + EGF-treated cells ( P < 0.01), d significant difference from U0124 + EGF-treated cells ( P < 0.05), e significant difference from U0124 + TNF-α + EGF-treated cells ( P < 0.001).

Article Snippet: For analysis of signaling pathways, cells were treated prior to addition of TNF-α or EGF with pharmacologic inhibitors including 2-[1-(3-dimethylaminopropyl)-1 H -indol-3-yl]-3-(1 H -indol-3-yl)-maleimide (10 μM bisindolylmaleimide [BIS] I, protein kinase C [PKC] inhibitor), or 2,3- bis (1 H -indol-3-yl)- N -methylmaleimide (10 μM BIS V, inactive analog of BIS I), 1,4-diamino-2,3-dicyano-1,4- bis (2-aminophenylthio)-butadiene (10 μM U0126, mitogen-activated protein kinase kinase 1 and 2 [MEK1/2] inhibitor; Promega, Madison, WI, USA), and 1,4-diamino-2,3-dicyano-1,4- bis (methylthio)-butadiene (10 μM U0124, inactive analog of U0126).

Techniques: Inhibition

Journal: Arthritis Research & Therapy

Article Title: Tumor necrosis factor alpha and epidermal growth factor act additively to inhibit matrix gene expression by chondrocyte

doi: 10.1186/ar1464

Figure Lengend Snippet: Inhibition of the mitogen-activated protein kinase pathway prevents tumor necrosis factor alpha (TNF-α) and epidermal growth factor (EGF)-mediated loss of aggrecan and type II collagen mRNA. Confluent chondrocytes were pretreated with U0124 (10 μm, inactive analog of U0126) or U0126 (10 μm, a MEK1/2 inhibitor), for 15 min, followed by treatment with TNF-α (30 ng/ml), EGF (10 ng/ml) or TNF-α + EGF for 24 hours. Levels of (a) aggrecan and (b) type II collagen mRNA were assessed by northern blot analysis of total RNA (10 μg). Levels were normalized to levels of 18S rRNA and data are expressed as the percentage of respective control ± standard error of the mean ( n = 5). a Significant difference from respective control ( P < 0.001), b significant difference from cultures treated individually with TNF-α or EGF ( P < 0.01), c significant difference from cultures treated with U0124 followed by addition of TNF-α + EGF ( P < 0.05).

Article Snippet: For analysis of signaling pathways, cells were treated prior to addition of TNF-α or EGF with pharmacologic inhibitors including 2-[1-(3-dimethylaminopropyl)-1 H -indol-3-yl]-3-(1 H -indol-3-yl)-maleimide (10 μM bisindolylmaleimide [BIS] I, protein kinase C [PKC] inhibitor), or 2,3- bis (1 H -indol-3-yl)- N -methylmaleimide (10 μM BIS V, inactive analog of BIS I), 1,4-diamino-2,3-dicyano-1,4- bis (2-aminophenylthio)-butadiene (10 μM U0126, mitogen-activated protein kinase kinase 1 and 2 [MEK1/2] inhibitor; Promega, Madison, WI, USA), and 1,4-diamino-2,3-dicyano-1,4- bis (methylthio)-butadiene (10 μM U0124, inactive analog of U0126).

Techniques: Inhibition, Northern Blot

Journal: European journal of immunology

Article Title: Rap1a activation by CalDAG-GEFI and p38 MAPK is involved in E-selectin-dependent slow leukocyte rolling

doi: 10.1002/eji.201041196

Figure Lengend Snippet: (A) Rolling velocity of WT neutrophils on E-selectin alone or E-selectin/ICAM-1 of either PLC inhibitor (U73122)- or DMSO-pretreated whole blood. (B) Rolling velocity of Rasgrp2−/− neutrophils on E-selectin alone or E-selectin plus ICAM-1 of either U73122- or DMSO-pretreated whole blood. (C) Rolling velocity of Rasgrp2−/− neutrophils on E-selectin alone or E-selectin/ICAM-1 of either p38 MAPK inhibitor (SB203580)– or DMSO-pretreated mice. Data presented as mean ± SEM from 3 mice. (D) (D) Whole human heparinized blood was treated with the p38 MAPK inhibitor SB203580 (10μM for 30 minutes at RT) or SB203580 (10μM) plus anti–LFA-1 antibody (10 μg/mL for 20 minutes at RT), then perfused through flow chambers coated with E-selectin with or without ICAM-1. Average rolling velocity of neutrophils on E-selectin (left) and E-selectin/ICAM-1 (right) is presented as mean ± SEM (n=3). (E) Whole human blood was treated with either Rap1-WT or Rap1-DN peptides (1μM for 30 minutes at RT), then perfused through flow chambers coated with E-selectin with or without ICAM-1. Average rolling velocity of neutrophils on E-selectin (left) and E-selectin/ICAM-1 (right) is presented as mean ± SEM (n=3). (F) HL-60 cells transfected with either siRNA specific for CalDAG-GEFI or a non-silencing control sequence were perfused through flow chambers coated with E-selectin and isotype antibody or KIM127 for 2 minutes at 5.94 dyn/cm2. The number of adherent cells per one representative field of view was determined. Data are from 3 experiments. #P < 0.05.

Article Snippet: For biochemical assays, bone-marrow derived neutrophils were isolated,[ 8 ] suspended in PBS (containing 1 mM each CaCl 2 and MgCl 2 ) and left untreated or were pretreated with a pharmacologic phospholipase C inhibitor ( {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 ; 1 μM; Cayman Chemical), a pharmacologic p38 MAPK inhibitor (SB203580; 10 μM; Biomol International) or DMSO control for 30 minutes Subsequently the cells were incubated under rotating conditions (65 rpm) for 10 minutes at 37°C on E-selectin coated dishes.

Techniques: Transfection, Control, Sequencing

Journal: European journal of immunology

Article Title: Rap1a activation by CalDAG-GEFI and p38 MAPK is involved in E-selectin-dependent slow leukocyte rolling

doi: 10.1002/eji.201041196

Figure Lengend Snippet: Bone marrow–derived neutrophils from WT mice (untreated or pretreated with different inhibitors: phospholipase C: U73122, p38 MAPK: SB203580), Pik3cg−/− mice, Plcg2−/− mice, and Rasgrp2−/− mice were plated on uncoated (unstimulated) or E-selectin–coated wells, and then lysates were prepared. (A-C) Total Rap1 and GTP-bound Rap1 protein levels were measured in untreated or pretreated neutrophils from WT mice (A, untreated or pretreated with a phospholipase inhibitor (U73122)), Plcg2−/− mice (B), and Rasgrp2−/− mice (C) after stimulation with E-selectin. Representative blots from 3 independent experiments are shown. (D) Total Rap1 and GTP-bound Rap1 protein levels were measured in unstimulated and stimulated neutrophils from WT mice after inhibiting p38 MAPK (I, SB203580, 10μM). Representative blots from 3 independent experiments are shown. (E) Total Rap1 and GTP-bound Rap1 protein levels were measured in unstimulated and stimulated neutrophils from Pik3cg−/− mice. Representative blots from 3 independent experiments are shown. (F+G) Bone-marrow-derived neutrophils from WT mice (untreated or pretreated with a phospholipase inhibitor (U73122)) and Rasgrp2−/− mice were plated in multiwell plates with or without E-selectin coating for 10 minutes, after which lysates were prepared and immunoblotted with antibody to phosphorylated p38 MAPK (phospho-p38) or total p38. Representative blots from 3 independent experiments are shown.

Article Snippet: For biochemical assays, bone-marrow derived neutrophils were isolated,[ 8 ] suspended in PBS (containing 1 mM each CaCl 2 and MgCl 2 ) and left untreated or were pretreated with a pharmacologic phospholipase C inhibitor ( {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 ; 1 μM; Cayman Chemical), a pharmacologic p38 MAPK inhibitor (SB203580; 10 μM; Biomol International) or DMSO control for 30 minutes Subsequently the cells were incubated under rotating conditions (65 rpm) for 10 minutes at 37°C on E-selectin coated dishes.

Techniques: Derivative Assay