Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 1. S1PR2 is increased in the serum and placental tissue of PE rats. (A) Measurement of S1P in serum via ELISA. (B) The expression of S1PR1, S1PR2 and S1PR3 in serum of control and PE rats was analyzed by reverse transcription‑quantitative PCR analysis. (C) The effect of S1P on the expression of S1PR2 in the plasma of PE rats was analyzed via ELISA. (D) The effect of S1P on the expression of S1PR2 in the placental tissues of PE rats was analyzed by western blotting. n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; #P<0.05 and ###P<0.001 vs. Model group; ∆P<0.05 and ∆∆∆P<0.001 vs. S1P group. S1P, sphingosine‑1‑phosphate; S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; ELISA, enzyme‑linked immunosorbent assay.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Control, Clinical Proteomics, Western Blot

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 2. Inhibition of S1PR2 with JTE‑013 decreases BP in PE rats. Effect of JTE‑013 on (A) SBP and (B) DBP in PE rats in tail‑cuff measurement. n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; #P<0.05, ##P<0.01 and ###P<0.001 vs. Model group; ∆P<0.05 vs. Model + S1PR2 antagonist low group. S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; BP, blood pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Inhibition, Control

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 3. Effect of JTE‑013 on serum NO and iNOS and the expression of eNOS in placental tissues. (A) Summarized data showing the inhibitory effect of JTE‑013 on serum NO levels in PE rats. (B) Summarized data showing the inhibitory effect of JTE‑013 on serum iNOS levels in PE rats. (C) Summarized data showing that JTE‑013 prevented the decreased expression of eNOS in placental tissues of PE rats. n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; ##P<0.01 and ###P<0.001 vs. Model group; ∆P<0.05 and ∆∆∆P<0.001 vs. Model + S1PR2 antagonist low group. S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; NO, nitric oxide.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Expressing, Control

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 4. JTE‑013 inhibits the PE model‑induced expression levels of VEGF and Flt‑1 receptor in PE rats. (A) Summarized data showing the inhibitory effect of JTE‑013 on the PE model‑induced mRNA levels of VEGF and Flt‑1 in reverse transcription‑quantitative PCR assay. (B) Representative western blotting images and summarized data showing the inhibitory effect of JTE‑013 on the PE model‑induced protein levels of VEGF and Flt‑1 in the placental tissues of PE rats. n=3. *P<0.05 and ***P<0.001 vs. Control group; #P<0.05, ##P<0.01 and ###P<0.001 vs. Model group; ∆∆P<0.01 and ∆∆∆P<0.001 vs. Model + S1PR2 antagonist low group. S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; VEGF, vascular endothelial growth factor; Flt‑1, fms‑like tyrosine kinase 1.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Expressing, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 5. JTE‑013 attenuates pathological changes in placental tissues and decreases inflammation in PE rats. (A) Summarized data showing the inhibitory effect of JTE‑013 on the increased expression of serum TNF‑α, IL‑1β and IL‑6, as determined by an enzyme‑linked immunosorbent assay. (B) Summarized data showing JTE‑013 attenuated the infiltration of inflammatory cells in placental tissues, as detected by hematoxylin and eosin staining (magnification, x100 or 200). n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; ##P<0.01 and ###P<0.001 vs. Model group; ∆P<0.05 and ∆∆P<0.01 vs. Model + S1PR2 antagonist low group. PE, preeclampsia; TNF‑α, tumor necrosis factor‑α; IL‑, interleukin; S1PR2, sphingosine‑1‑phosphate receptor 2; lab, labyrinth; JZ, junctional zone; de, decidua.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Cell reports

Article Title: HIV-1 Vpr drives a tissue residency-like phenotype during selective infection of resting memory T cells.

doi: 10.1016/j.celrep.2022.110650

Figure Lengend Snippet: Figure 4. Incoming Vpr is sufficient to drive TRM induction in resting memory CD4+ T cells (A) CD69 (left) and CD69/CXCR6 (right) co-expression in response to IL-7 in the presence of integrase inhibitor raltegravir (n = 6). (B) Quantification of integrated provirus and 2LTR circles in FACS-sorted target CD4+ memory T cells after 72 h of cell-to-cell spread in the presence or absence of raltegravir. (C) Schematic depicting the viruses and VLPs used in (D–H). (D) Western blot showing Vpr packaging into virions of HIV-1 WTPM, VprPM, WT, DVpr, and DVpr complemented with FLAG-tagged Vpr in trans (DVpr+Vprtrans). (E) CD69 (left) and CD69/CXCR6 (right) upregulation in response to IL-7 on Gag+ resting memory CD4+ T cells at 72 h infected with the indicated HIV-1 viruses (n = 8). (F) Expression of CD69 (left) and CD69/CXCR6 (right) in response to IL-7 on Gag+ resting memory CD4+ T cells at 72 h post spinoculation of HIV-1 WT, Vpr, and DVpr+Vprtrans (n = 10). (G) Western blot showing packaging of FLAG-tagged Vpr into Env-VLPs or full-length HIV-1 WT or DVpr. (H) Expression of CD69 (left) and CD69/CXCR6 (right) in response to IL-7 on Gag+ resting memory CD4+ T cells at 72 h post spinoculation of Env-VLPs with or without Vpr (n = 5). All measurements were made after 72 h or at the indicated time post co-culture or spinoculation. Data are the mean ± SEM. Paired two-tailed t test or one-way ANOVA with Bonferroni or Dunnett’s post test was used. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant. EV, empty vector.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER HIV-1 pNL4.3 Vpr S79A A. Reuschl This study HIV-1 pNL4.3 Vpr R80A A. Reuschl This study Biological samples PBMCs isolated from buffy coats from healthy donors UK NHS Blood and Transplant Service N/A Tonsillar tissue from elective tonsillectomy Imperial College Infectious Diseases Biobank N/A Lymph nodes obtained from field surgery of participants undergoing surgery for diagnostic purposes and/or complications of inflammatory lung disease University of KwaZulu-Natal N/A Human Serum from humanmale AB plasma Sigma-Aldrich Cat# H4522-20ML Chemicals, peptides, and recombinant proteins Phytohemagglutinin-L (PHA-L) Sigma Cat# 11249738001 Interleukin-2 (Human, rDNA derived) CFAR Cat# 86/500 Fugene 6 Transfection Reagent Promega Cat# E2691 DNase I Sigma Cat# DN25-100MG CellTraceTM Far Red Cell Proliferation Kit ThermoFisher Cat# C34564 Fixable Viability Dye eFluorTM 450 ThermoFisher Cat# 65-0863-14 Recombinant human IL-7 Miltenyi Biotec Cat# 130-095-362 Recombinant Human IL-15 Peptrotech Cat# 200-15 Recombinant Human IL-12 p70 Peprotech Cat# 200-12 Recombinant Human TGF-b1 Peprotech Cat# 100-21C T20 CFAR Cat# 0984 Efavirenz CFAR Cat# 0977 Raltegravir CFAR Cat# 0980 Ruxolitinib Selleckchem Cat# S1378 Zombie NIRTM Fixable Viability Kit Biolegend Cat# 423106 Zombie UVTM Fixable Viability Kit Biolegend Cat# 423108 Zombie AquaTM Fixable Viability Kit Biolegend Cat# 423102 Super Bright Staining Buffer ThermoFisher Cat# SB-4400 Brefeldin A Solution Biolegend Cat# 420601 Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich Cat# P1585-1MG Ionomycin Sigma-Aldrich Cat# I9657-1MG Intracellular Staining Permeabilization Wash Buffer Biolegend Cat# 421002 FOXP3 Fix/Perm Buffer Set Biolegend Cat# 421403 True-PhosTM Perm Buffer Biolegend Cat# 425401 RLT Buffer (RNeasy Lysis Buffer) Qiagen Cat# 79216 b-mercaptoethanol (Sigma-Aldrich) Sigma-Aldrich Cat# M3148 Phusion Hot Start Flex DNA Polymerase NewEngland Biolabs Cat# M0535L TaqManTM Master-Mix ThermoFisher Cat# 4369016 SuperScriptTM IV Reverse Transcriptase ThermoFisher Cat# 18090050 Fast SYBRTM Green Master Mix Applied Biosystems Cat# 4385612 Hoechst33342 ThermoFisher Cat# H3570 Critical commercial assays MojoSortTM Human CD4 T Cell Isolation Kit Biolegend Cat# 480010 CD45RA MicroBeads, human Miltenyi Biotec Cat# 130-045-901 (Continued on next page) Cell Reports 39, 110650, April 12, 2022 e3

Techniques: Expressing, Western Blot, Infection, Co-Culture Assay, Two Tailed Test, Plasmid Preparation

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 4. The effects of milk-derived extracellular vesicles (MEVs) supplementation on baseline homeostatic human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148-5P transcript levels (a), DNMT1 protein abundance (b) and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (d), and DNMT enzymatic activity and DNMT1 protein level (e). * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 6. The effects of milk-derived extracellular vesicles (MEVs) on IFN-γ primed human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (p < 0.05) (d), and DNMT enzymatic activity and DNMT1 protein level (p < 0.05) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 7. The effects of milk-derived extracellular vesicles (MEVs) on primed human microglia clone 3 (HMC3) cells 9 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels relative to miR-148a-5P levels (p ≤ 0.05) (d), and DNMT enzymatic activity relative to DNMT1 protein level (A.U) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Journal of cell science

Article Title: Tbl1 promotes Wnt-β-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

doi: 10.1242/jcs.261241

Figure Lengend Snippet: Fig. 1. Overexpression of Tbl genes decreases Tcf7l1 protein levels and is beneficial for maintaining the stemness of mESCs. (A) Western blot analysis of Tcf7l1 protein levels in cells overexpressing Flag tag alone (empty vector control, Flag) or Flag–Tbl1. (B) Densitometric analysis of the relative protein levels of Tcf7l1, as shown in A, was performed with ImageJ software. Protein levels were normalized to those of β-actin. (C) Western blot analysis of Tcf7l1 levels in cells overexpressing Flag tag alone (Flag) or Flag–Tblr1. (D) Densitometric analysis of the relative protein level of Tcf7l1, as shown in C, was performed with ImageJ software. Protein levels were normalized to those of β-actin. (E) RT–qPCR analysis of Tcf7l1 levels in cells overexpressing Flag tag alone (Flag) or Flag–Tbl1. (F) RT–qPCR analysis of Tcf7l1 levels in cells overexpressing Flag tag alone (Flag) or Flag–Tblr1. (G) AP staining of mESCs expressing Flag tag alone, Flag–Tbl1 or Flag–Tblr1 cultured in serum-containing medium without LIF for 8 days. Scale bars: 100 μm. (H) Quantification of AP-positive colonies as shown in G. (I) RT–qPCR analysis of the expression of Oct4, Sox2, Klf4, Esrrb, Nanog and Gata4 in mESCs expressing Flag tag alone, Flag–Tbl1 or Flag–Tblr1. (J) Left: AP staining of mESCs expressing either Flag tag alone or Flag–Tbl1 and transfected with a plasmid encoding HA– Tcf7l1. Cells were cultured in medium containing LIF and serum. Right: immunofluorescence (IF) of HA–Tcf7l1 mESCs transfected with Flag or Flag–Tbl1 showing Oct4 (red) and Hoechst 33342 (blue). IF images are representative of three experiments. Scale bars: 100 μm. (K) Quantification of AP-positive colonies as shown in J. All quantitative data are presented as mean±s.d. of n=3 biological replicates. *P<0.05, **P<0.01 versus Flag, as determined by unpaired, two-tailed Student’s t-test (B,D–F,K) or one-way ANOVA with Sidak’s multiple comparisons test (H,I).

Article Snippet: The primary antibodies used were specific for Flag (F1804, Sigma-Aldrich; 1:2000), Tcf7l1 (bs-12891, Bioss; 1:1000), HA (3724, Cell Signaling Technology; 1:1000), β-actin (66009-1-Ig, Proteintech; 1:1000), β-tubulin (66240-1-Ig, Proteintech; 1:1000), Oct4 (SC-5279, Santa Cruz; 1:1000), Sox2 (66411-1-Ig, Proteintech; 1:1000), Klf4 (381633, ZENBIO; 1:1000), Tbl1 (823294, ZENBIO; 1:1000), Tblr1 (R383062, ZENBIO; 1:1000), non-phospho (Active) β-catenin (Ser33/37/Thr41) (8814, Cell Signaling Technology; 1:1000), β-catenin (SC-7199, Santa Cruz; 1:1000) and ubiquitin (SC-8017, Santa Cruz; 1:1000).

Techniques: Over Expression, Western Blot, FLAG-tag, Plasmid Preparation, Control, Software, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Transfection, Immunofluorescence, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 1. RASEF expression in tumor tissues and cell lines. A, expression of RASEF in 12 clinical lung cancers (T; 4 clinical lung ADC, 4 clinical lung SCC, and 4 clinical SCLC) and corresponding normal lung tissues (N) detected by semiquantitative RT-PCR analysis. B, expression of RASEF in 22 lung cancer cell lines and a bronchial epithelial cell line BEAS-2B detected by semiquantitative RT-PCR analysis. ASC indicates lung adenosquamous cell carcinoma; LCC, large cell carcinoma. C, Western blot analysis of RASEF protein using anti-RASEF antibody. IB, immunoblotting. D, expression and subcellular localization of endogenous RASEF protein in RASEF-positive and RASEF-negative lung cancer cell lines, and bronchial epithelial cells. RASEF was stained mainly at the cytoplasm in A549 and NCI-H2170 cells, whereas no staining was observed in DMS114 and bronchial epithelia–derived BEAS-2B cell lines.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Derivative Assay

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 4. Enhanced phosphorylation of ERK1/2 by RASEF in lung cancer cells. A, expression of MAPK signal molecules and their phosphorylation levels in DMS114 cells transfected with RASEF expression vector or mock plasmid. B, expression of MAPK signal molecules and their phosphorylation levels in NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC). C and D, expression levels of downstream target genes of MAPK cascade were regulated by RASEF expression in lung cancer cells. Total RNA from BEAS-2B and DMS114 cells transfected with RASEF expression vector or mock plasmid (C) and A549 and NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC; D) were subjected to reverse-transcription reaction, followed by PCR reaction to evaluate the expression levels of CCND1, CCNB1, and CDKN1A transcription. Western blotting with antiphosphorylated ERK1/2 antibody was conducted to confirm the change of ERK1/2 phosphorylation according to RASEF expression.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Phospho-proteomics, Expressing, Transfection, Plasmid Preparation, Control, Reverse Transcription, Western Blot

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 5. Identification of ERK1/2-interacting sites on RASEF. A, interaction of endogenous RASEF with endogenous ERK1/2. The immunoprecipitates obtained using anti-RASEF antibody were subjected to Western blotting with anti-ERK1/2 antibody. B, schematic representation of various partial constructs of RASEF expression vector. C and D, determination of the ERK1/2-interacting regions on RASEF by immunoprecipitation experiments using DMS114 cells transfected with vectors expressing partial RASEF protein. COOH-terminal part of RASEF (codons 520–575) was likely to be ERK1/2-interacting region.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Western Blot, Construct, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection

Journal: Cell stem cell

Article Title: ROS are required for mouse spermatogonial stem cell self-renewal.

doi: 10.1016/j.stem.2013.04.001

Figure Lengend Snippet: Figure 7. Regulation of GSC Proliferation by p38 MAPK and JNK (A) Western blot analyses of GSCs cultured with H2O2. (B and C) Western blot analyses of p38 MAPK (B) and JNK (C) phosphorylation after cytokine treatment. Cells were starved for 4 days, and the samples were collected 4 hr after cytokine addition. (D) Western blot analyses of p38 MAPK and JNK phosphorylation after ROS depletion. Cells were cultured for 6 days with FGF2 and GDNF. Apoc- ynin was added on the second day after plating, whereas LA and DPI were added on the fourth and fifth days, respectively. (E) Appearance of GSCs after the addition of SB203580 or SP600125. Cells were cultured for 6 days with the indicated inhibitors. (F) Inhibition of GSC proliferation by SB203580 or SP600125. Cells were cultured for 6 days with the indicated inhibitors (n = 6). The results of two experiments are shown. (G) RT-PCR analyses of Nox gene expression. Cells were cultured for 2 days with the indicated inhibitors. (H) Macroscopic appearance of recipient testes that received SB203580- or SP600125-treated GSCs. (I and J) Colony count (I) and total increase in SSC number (J) after SB203580 or SP600125 treatment (n = 13 for control and SB203580, n = 16 for control and SP600125). The results of two (SB203580) or three (SP600125) experiments are shown. The scale bars represent 100 mm (E) and 1 mm (H). The error bars represent SEM. See also Tables S1 and S2.

Article Snippet: LA (2 mM), DPI (1 mM; both from Sigma-Aldrich, St. Lois, MO, USA), apocynin (1 mM; Tokyo Chemical Industry, Tokyo, Japan), SP600125 (40 mM), and SB203580 (30 mM; both from Selleck Chemicals, Houston, TX, USA) were added to the cultures at the time of plating in order to examine their effects on cell proliferation.

Techniques: Western Blot, Cell Culture, Phospho-proteomics, Inhibition, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control

Journal: Free radical biology & medicine

Article Title: Lafora disease fibroblasts exemplify the molecular interdependence between thioredoxin 1 and the proteasome in mammalian cells.

doi: 10.1016/j.freeradbiomed.2013.07.001

Figure Lengend Snippet: Fig. 1. Measurement of Trx1 mRNA and protein levels during cell proliferation in NIH3T3 fibroblasts. (A) DNA synthesis in NIH3T3 fibroblasts measured by the BrdU incorporation assay (Roche). Results of BrdU incorporation are the means and SD from two separate experiments with triplicate samples, and Trx1 mRNA levels represent the means and SD from two separate experiments with duplicate samples. *po0.05, statistically significant difference from control value. (B) Trx1 mRNA levels measured at various times of NIH3T3 cell culture by RT-PCR. (C) Trx1 protein levels determined by Western blot at various times of NIH3T3 cell proliferation using anti-Trx1 antibody. α-Tubulin was used as a loading control.

Article Snippet: Membranes were blocked with 0.05 g/ml nonfat milk or with 0.05 g/ml bovine serum albumin (BSA) in Tris-buffered saline containing 0.2% Tween 20 (TBST), depending on the antibody, washed three times at room temperature, and incubated for 2 h at room temperature with primary antibodies diluted in TBST with 0.01 g/ml nonfat milk as follows: antioxidant enzymes as previously used [26], Trx1 (1:1000, Abcam, Cambridge, MA, USA), TrxR1 (1:1000, Abcam), Trx2, and Bax (1:1000, GeneTex); ubiquitin–protein conjugates (1:1000, Enzo Life Sciences); PDI (1:1000, Cell Signaling); BIP (1:1000, Cell Signaling); ATF4 (1:1000, Santa Cruz Biotechnologies); CHOP (1:1000, Santa Cruz Biotechnologies); and proteasome 20 S α7 subunit (1:1000, Enzo Life Sciences). α-Tubulin or β-actin antibodies (1:1000, Santa Cruz Biotechnologies) were used as loading controls.

Techniques: DNA Synthesis, BrdU Incorporation Assay, Control, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The Journal of investigative dermatology

Article Title: Low herpesvirus entry mediator (HVEM) expression on dermal fibroblasts contributes to a Th2-dominant microenvironment in advanced cutaneous T-cell lymphoma.

doi: 10.1038/jid.2011.470

Figure Lengend Snippet: Figure 2. CXC chemokine ligand (CXCL)9, CXCL10, and CXCL11 expression induced by LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes) via phosphorylation of inhibitor jBa (IjBa). (a) Human dermal fibroblasts were cultured with IFN-g (10 ng ml1) or LIGHT (100 ng ml1) or their combination. IkBa phosphorylation was detected in fibroblasts stimulated with LIGHT, but not with IFN-g alone. A representative picture of three experiments is shown. (b, c) Human dermal fibroblasts were cultured with medium only or with IFN-g (10 ng ml1) or LIGHT (1, 10, and 100 ng/ml1) or their combination for 16 hours after pre-incubation with medium or sc-514 (20 and 50 mM). (b) Quantitative reverse transcriptase (RT)–PCR was performed to measure CXCL9, CXCL10, and CXCL11 expression relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (c) CXCL9, CXCL10, and CXCL11 levels in the culture supernatants were measured. Data are presented as mean±standard deviation. *Po0.05. Results are representative of three experiments.

Article Snippet: After transfer to nitrocellulose membrane (Invitrogen), the membrane was blotted with mouse anti-human phospho-IkBa mAb (Cell Signaling Technology, Boston, MA), mouse anti-human IkBa mAb (Cell Signaling Technology), or mouse anti-human GAPDH mAb overnight at 4 1C and with the appropriate secondary antibody for 1 hour at room temperature.

Techniques: Expressing, Virus, Phospho-proteomics, Cell Culture, Incubation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Standard Deviation