Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Vitamin E relieves chronic obstructive pulmonary disease by inhibiting COX2-mediated p-STAT3 nuclear translocation through the EGFR/MAPK signaling pathway.

doi: 10.1038/s41374-021-00652-z

Figure Lengend Snippet: Fig. 2 Vitamin E downregulates EGFR and inactivates MAPK signaling pathway. A Venn diagram of the intersection of drug and disease targets. B Network diagram of gene-drug-disease interaction relationship. C KEGG enrichment analysis results of 30 candidate targets. D Box diagram of EGFR expression in COPD attained through microarray dataset GSE3320, wherein blue box on the left indicates normal tissue samples and red box on the right indicates COPD samples. E EGFR expression in lung tissues of rats after CS or vitamin E treatment was detected by immunohistochemistry. F EGFR, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2 protein expression in lung tissues of rats after CS or vitamin E treatment was detected by Western blot. G Statistical diagram of Panel F. The data in the figure are measurement data, which are expressed by the mean ± standard deviation. The data of each group are analyzed by unpaired t-test, and the data among multiple groups are compared by one-way ANOVA and Tukey’s post hoc test. *p < 0.05 vs. the control group, #p < 0.05 vs. the CS group, n = 6.

Article Snippet: Then the membrane was probed with primary antibodies against COX2 (1: 1000, ab179800, Abcam), EGFR (1:2000, ab52894, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab76315, Abcam), p38 (1:1000, 9212, Cell Signaling Technologies [CST], Beverly, MA,USA), p-p38 (1:1000, 9211, CST), JNK (1:2000, 9252, CST), p-JNK (1:2000, 9255, CST), ERK1/2 (1:1000, 4695, CST), p-ERK1/2 (1:1000, 4370, CST), and GAPDH (1:5000, ab8254, Abcam) overnight at 4 °C.

Techniques: Expressing, Microarray, Immunohistochemistry, Western Blot, Standard Deviation, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Vitamin E relieves chronic obstructive pulmonary disease by inhibiting COX2-mediated p-STAT3 nuclear translocation through the EGFR/MAPK signaling pathway.

doi: 10.1038/s41374-021-00652-z

Figure Lengend Snippet: Fig. 3 Vitamin E relieves CS-induced HBE cell damage. A RT-qPCR was used to assess the expression of EGFR in HBE cells of each group. B Western blot was used to assess the expression of EGFR, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2 in HBE cells of each group. C Statistical map of Panel B. D CCK8 assay was used to detect the viability of HBE cells in each group. E Annexin V/PI staining was used to detect the apoptosis of HBE cells in each group. F ELISA was used to detect the expression of inflammatory factors in the supernatant of HBE cells of each group. G ROS level in HBE cells of each group. H The SOD activity and MDA content in HBE cells of each group. The data in the figure were all measurement data, expressed as the mean ± standard deviation. The data of each group were analyzed by unpaired t-test, and the comparison of data among multiple groups was performed by one-way ANOVA and Tukey’s post hoc test. *p < 0.05 vs. the Vector group, #p < 0.05 vs. the vector + CS group, and $p < 0.05 vs. the vector + CS + vitamin E group.

Article Snippet: Then the membrane was probed with primary antibodies against COX2 (1: 1000, ab179800, Abcam), EGFR (1:2000, ab52894, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab76315, Abcam), p38 (1:1000, 9212, Cell Signaling Technologies [CST], Beverly, MA,USA), p-p38 (1:1000, 9211, CST), JNK (1:2000, 9252, CST), p-JNK (1:2000, 9255, CST), ERK1/2 (1:1000, 4695, CST), p-ERK1/2 (1:1000, 4370, CST), and GAPDH (1:5000, ab8254, Abcam) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation, Comparison, Plasmid Preparation

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 3 Validation of cba-miR-222-3p targeting TRAF7 and TRAF7 expression in the testes of striped hamsters. (a) Sequences and peak maps of cba-miR-222-3p, TRAF7-WT, and TRAF7-MT. (b) Relative luciferase activity detected by Dual-Luciferase Reporter Assay. (c) Immunohistochemistry (IHC, tissue microarray [TMA]) of TRAF7 in testes. (d) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (e) Protein expression levels of TRAF7 in the testes detected by western blot (n = 4). (f) Pearson correlation analysis of cba-miR-222-3p and TRAF7. LD, long daylength; MD, moderate daylength; SD, short daylength; ∗, P < 0.05; ∗∗, P < 0.01.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Biomarker Discovery, Expressing, Luciferase, Activity Assay, Reporter Assay, Immunohistochemistry, Microarray, Western Blot

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 4 Expression of cba-miR-222-3p and TRAF7 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis after in vivo injection in the testes of striped hamsters. (a) Schematic diagram of in vivo injection experiment of miRNA mimics. (b) Fluorescence in situ hybridization (FISH, tissue microarray [TMA]) of cba-miR-222-3p in testes after in vivo injection. (c) Fluorescence intensity of cba-miR-222-3p detected by FISH (TMA; n = 4). (d) Immunohistochemistry (IHC, TMA) of TRAF7 in testes after in vivo injection. (e) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (f) TUNEL (TMA) staining of the testes after in vivo injection. (g) The proportion of TUNEL (TMA) staining with apoptotic activity in the testes (n = 4). (h) Relative expression of MEKK3 (n = 6). (i) Relative expression of p38 (n = 6). (j) Relative expression of p53 (n = 6). AG, agomir injection; NC, agomir negative control injection; DAPI, 4′6′-diamidino-2-phenylindole. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Expressing, TUNEL Assay, In Vivo, Injection, Fluorescence, In Situ Hybridization, Microarray, Immunohistochemistry, Staining, Activity Assay, Negative Control

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 1. Id1 Is a Target of the TbRI Inhibitor in PCTCs and in a Patient-Derived Mouse Model for Glioma (A) The genes were regulated by the treatment with 2 mM TbRI inhibitor for 3 hr in 11 human glioma PCTCs with a fold change over 1.4 or below 0.6 and a p < 0.001. (B) ID3, ID1, Smad7, and RhoB transcript levels were determined by qRT-PCR analysis. GAPDH RNA levels were used as an internal normalization control. *p < 0.05; **p < 0.001. Data are presented as means ± SD. (C and D) Cells from GBM1 and GBM2 neuro- spheres were inoculated in the brain of immuno- compromised mice. Twenty days after surgery, mice were orally treated twice a day with 100 mg/kg of TbRI inhibitor for 30 days. (C) Tumor area was quantified. (D) Images from the entire mouse brains were obtained by MRI (arrowheads indicate tumors). (E) Immunohistochemistry for Id1 (upper panel) and double immunofluorescence for Id1 and CD31 (middle panel) were performed in tumors generated by GBM neurospheres. For immunoflu- orescence, nuclei were counterstained with Hoechst. Scale bar, 100 mm. Representative images are shown and percentage of Id1-positive cells was calculated (lower panel). Data are pre- sented as means ± SD. (F) Cells from GBM1 neurospheres were inocu- lated in the brain of immunocompromised mice. Forty days after surgery, mice were orally treated twice a day with 100 mg/kg of TbRI inhibitor for 10 days. Subsequently, 10 mm slides from mouse brains were stained with H&E and tumors were localized and microdissected. ID1 tran- script levels were determined by qRT-PCR (n = 4 for vehicle; n = 3 for TbRI inhibitor). Data are presented as means ± SD. See also Fig- ure S1.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Derivative Assay, Quantitative RT-PCR, Control, Immunohistochemistry, Generated, Staining

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 4. Patient-Derived Neurospheres Have a CD44high/Id1high Cell Population (A) Cells from GBM1 neurospheres were dissociated and seeded on coverslips. Immunocytochemistry for Id1and CD44 was performed and nuclei were counter- stained with Hoechst. Scale bar, 10 mm. Quantification of Id1 and CD44 expression levels per cell is shown. (B) FACS analysis of CD44 levels was performed in GBM neurospheres (upper panel). Isotype control is shown. Cells with high or low levels of CD44 from the indicated GBM neurospheres were sorted by FACS and CD44, ID1, ID2, and ID3 transcript levels were determined by qRT-PCR (lower panels). *p < 0.01; **p < 0.001 compared to CD44low. Data are presented as means ± SD. (C) Cells from GBM1 neurospheres were sorted by FACS according to CD44 levels, and the levels of Id1, Id3, and tubulin were determined by immunoblotting. See also Figure S4.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Derivative Assay, Immunocytochemistry, Staining, Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 6. Id Proteins Mediate the Effect of TGF-b on the CD44high Population and the Oncogenic Potential of GBM Neurospheres (A) Cells from GBM1 were infected with the indicated lentivirus. Neurospheres were treated with 100 pM TGF-b1 for 3 hr, 2 mM TbRI inhibitor for 8 hr, or left untreated, and the levels of Id1, Id3, p-Smad2, Smad2, and tubulin were determined by immunoblotting. (B) Cells from GBM1 neurospheres previously infected with the indicated lentivirus were treated with 100 pM TGF-b or 2 mM TbRI inhibitor for 7 days or left untreated. FACS analysis of CD44 levels (left panel) and quantification of the frequency of CD44high cells are shown (right panel). Bars represent the percent of CD44high cells in untreated, TGF-b-treated, and TbRI inhibitor-treated lentiviral-infected neurospheres, as indicated. Data are presented as means. (C–E) Cells from GBM1 neurospheres previously infected with the indicated lentivirus were treated for 7 days with 2 mM TbRI inhibitor, or left untreated. Subse- quently, cells were dissociated, counted, and equal numbers of cells were inoculated in the brain of NOD-SCID mice. (C) Images from the entire mouse brains were obtained by MRI. Arrowheads indicate tumors. (D) Tumor area was quantified (p = 0.004 comparing mice inoculated with untreated neurospheres with mice inoculated with neurospheres treated with the TbRI inhibitor; p = 0.002 comparing mice inoculated with neurospheres infected with the lentivirus control with mice inoculated with neurospheres infected with the sh2 ID1/ID3). (E) Tumor incidence was determined. Data are presented as means ± SD. (F–H) Cells from GBM1 neurospheres previously infected with the indicated lentivirus were treated during 7 days with 2 mM TbRI inhibitor or left untreated. Subse- quently, cells were dissociated, counted, and equal numbers of cells were inoculated in the brain of immunocompromised mice. (F) Images from the entire mouse brains were obtained by MRI. Arrowheads indicate tumors. (G) Tumor area was quantified (p = 0.04 comparing mice inoculated with control neurospheres with mice inoculated with Id1 overexpressing neurospheres), and (H) tumor incidence was determined. Data are presented as means ± SD. See also Figure S6.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Infection, Western Blot, Control

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 7. TbRI Inhibitor Decreases Id1, Id3, and CD44 Levels in Tumors and Prevents Tumor Recurrence in Vivo (A) Scheme showing the experimental procedure. (B and C) Cells from GBM1 neurospheres were inoculated in the brain of immunocompromised mice. Forty days after surgery, mice were orally treated twice a day with 100 mg/kg of TbRI inhibitor for 10 days. Subsequently, human tumor cells were isolated from the brain of mice treated or untreated with TbRI inhibitor through sorting of human MHC-I-positive cells. (B) ID1, ID3, and CD44 transcripts levels were determined by qRT-PCR, and (C) CD44 levels were assessed by FACS. Isotype control is shown. Data are presented as means ± SD. (D and E) Human tumor cells obtained from mice treated or untreated with the TbRI inhibitor were inoculated in the brain of secondary mice. (D) Thirty days after surgery, images from the entire mouse brains were obtained by MRI, tumor area was quantified (n = 4 mice inoculated with cells from untreated mice; n = 8 mice inoculated with cells from TbRI inhibitor treated mice), and (E) tumor incidence was determined. Data are presented as means ± SD.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: In Vivo, Isolation, Quantitative RT-PCR, Control

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 8. Id1 and CD44 Coexpression Correlates with Overall Survival in GBM Patients (A) A tissue microarray of 43 different GBM patients was stained with an antibody against Id1 and the frequency of Id1-positive nuclei was calculated. (B) CD44/Id1 and CD31/Id3 double immunofluorescence of samples from GBM patients. Magnification of the indicated areas stained with Id1/CD44 and Id3/ CD31 are shown in the right panels. Nuclei were counterstained with Hoechst. Scale bar, 50 mm. (C) CD44, Id1, and CD31 immunohistochemistry of samples from GBM patients. Scale bar, 100 mm. (D) Kaplan-Meier curves showing that the overall survival of patients with both ID1 mRNA levels upregulated R3-fold and CD44 mRNA levels upregulated R10-fold is significantly lower than the rest of the patients (p = 0.03) by log-rank test. Data obtained from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) program form the National Cancer Institute. See also Figure S7.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Microarray, Staining, Immunohistochemistry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 2. Characteristics of the HHR thymus and differentiation status of thymocytes in the thymus. (A) Graphs show the weights and cellularities of SDR and HHR thymi at 4–5 wk of age. Three SDRs and HHRs were used for each analysis. Data are shown as mean 6 SD. *p , 0.05. (B) Histological sections of thymi from 4-wk-old SDRs and HHRs were stained with H&E. Scale bars represent 1 mm. Representative results of two SDRs and HHRs are shown. (C) The differentiation status of thymocytes was analyzed with a flow cytometer. Representative results of three SDRs and HHRs are shown. An arrow indicates a population of DP thymocytes with decreased CD4 levels. Averaged values from three independent flow cytometric analyses for the proportions of DP, CD4-SP, CD8-SP, and DN thymocytes are shown in the lower graph. Data are shown as mean 6 SD. (D) Expression levels of Cd4 and Cd8 genes in the thymus were analyzed by real-time RT-PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Staining, Cytometry, Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 3. nTreg numbers in the HHR thymus. (A) Expression levels of Cd25 and Foxp3 genes in CD4-SP thymocytes were analyzed by real-time RT- PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) Upper, The proportion of CD4+CD25+ cells in the thymus was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of CD4+CD25+ cells are shown in the graph. Data are shown as mean 6 SD. Lower, The proportion of Foxp3+ cells in the CD4+CD25+ cell fraction was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of Foxp3+ cells in the CD4+CD25+ cell fraction are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. (C) Upper, Estimated values for the proportion of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of Foxp3+ cells in the CD4+CD25+ cell fraction obtained from flow cytometric analysis and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. Lower, Estimated values for the absolute number of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of total thymus cell number (Fig. 2A) and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 5. Loss of Ly49s3 gene expression in HHR thymic cDCs. (A) Left, Genome-wide microarray CGH analysis, performed with genomic DNA from SDR and HHR livers, shows the deletion of four Ly49 family genes—Ly49s4, Ly49i4, Ly49s3, and Ly49i3—in chromosome 4 at the q42 region (shaded area). Data shown are representative of two independent analyses. Right, Genomic PCR with DNA from SDR and HHR livers was performed to confirm the deletion of DNA in this region. As an internal standard, the Ccr4 gene, located at chromosome 8q32, was used. (B) Left, RT-PCR analysis of the expression of the Ly49s3 gene was performed with total RNA from SDR and HHR thymi. As an internal standard, the Gapdh gene was used. Middle, RT- PCR analysis of Ly49s3 gene expression in DP, CD4-SP, and CD8-SP thymocytes of the SDR thymus was performed with total RNA from the cells. Note that it was not possible to isolate pure DN thymocytes by positive and/or negative selection using CD4 and CD8a microbeads because the remaining cells after the selection of DP, CD4-SP, and CD8-SP cells are a mixture of DN thymocytes and all of the other types of cells. Right, RT-PCR analysis of Ly49s3 gene expression in cDCs of SDR and HHR thymi was performed with total RNA from the cells.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Gene Expression, Genome Wide, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Selection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 6. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture. (A) A total of 1 3 106 CD4-SP thymocytes isolated from the SDR thymus were cultured with 2.5 3 105 cDCs from the SDR thymus, and 1 3 106 CD4-SP thymocytes from the HHR thymus were cultured with 2.5 3 105 cDCs from the HHR thymus. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) To confirm the expression levels of Foxp3 and CD25, an additional mixed-cell culture experiment was performed, and the proportion of Foxp3+ or CD25+ cells was determined by flow cytometric analysis. (C) As control experiments, 1 3 106 CD4-SP thymocytes were cultured alone for 3 d and the same real-time RT-PCR analyses as above were performed. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for SDR cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (D) Effects of cDCs on nTreg marker gene expression. The expression levels of nTreg marker genes in mixed-cell (A) and control cultures (C) are shown in the same graph relative to the value for the SDR control culture, which is set at 1. Statistical analysis was performed between the induction ratios of gene expression. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 1 3 106 CD4+CD82CD252 thymocytes isolated from SDR and HHR thymi were cultured with 2.5 3 105 cDCs isolated from SDR and HHR thymi, respectively. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Isolation, Quantitative RT-PCR, Control, Gene Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 7. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture using Ly49s3-expressing HHR thymic cDCs. (A) The FLAG- tagged Ly49s3 structure is schematically represented. R: arginine residue. (B) Left, 293T cells were transfected with recombinant vectors before being packaged into the virus, and cell lysates were subjected to Western blot analysis with the anti-FLAG M2 Ab. Right, The proteins on the membrane were stained with fast green. (C) Left, cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3, and the expression of the fusion protein on the surface of cDCs was confirmed by fluorescence microscopic observations with the anti-FLAG M2 Ab. Right, Phase contrast appearance of the identical cells shown in the left photographs. Note dendrites on the surface of the cells. The cells were suspended in buffer and all the photos were taken. Original magnification 3800. (D) A total of 2.5 3 105 cDCs isolated from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 (Ly) or the mock vector (Mo) and then mixed with 1 3 106 CD4-SP thymocytes isolated from the HHR thymus. At 3 d later, total RNAwas extracted and the expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture, using cDCs transduced with the mock vector, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 2.5 3 105 cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 and then mixed with 1 3 106 CD4-SP thymocytes from the HHR thymus. Next 5 mg of anti-rat MHC class I Ab or normal IgG was added to the culture. At 3 d later, total RNA was extracted, and the expression levels of nTreg marker genes and MHC class II genes were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture in the presence of normal IgG, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (F) Summary of the experiments performed using the lentiviral vector of the Ly49s3 gene and anti-MHC class I Ab. The level of each gene is shown relative to the value for mock-transduced cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Residue, Transfection, Recombinant, Virus, Western Blot, Membrane, Staining, Transduction, Plasmid Preparation, Isolation, Quantitative RT-PCR

Journal: Cancer science

Article Title: Activation of epidermal growth factor receptor signaling by the prostaglandin E(2) receptor EP4 pathway during gastric tumorigenesis.

doi: 10.1111/j.1349-7006.2011.01847.x

Figure Lengend Snippet: Fig. 1. Gene expression levels of epidermal growth factor receptor (EGFR) ligands, EGFR family members (a) and a disintegrin and metalloproteinases (ADAMs) (b) in the stomach of the respective models (mean ± SD) calculated from microarray results. Asterisks indicate P < 0.05 versus the wild-type level. (c) Fluorescence immunostaining for phosphorylated EGFR at Tyr845 (green) in the gastric mucosa of the indicated genotype mice. DAPI staining for nuclei is visualized in red. Bars indicate 100 lm.

Article Snippet: Antibody for phosphorylated EGFR (Tyr845) (cell signaling), Ki-67 (DakoCytomation, Carpenteria, CA, USA), or active b-catenin (Millipore, Billerica, MA, USA) was used as the primary antibody.

Techniques: Gene Expression, Microarray, Fluorescence, Immunostaining, Staining

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Fold changes in the microarray for all 35 genes were found to be differentially regulated by C. pneumoniae infection and satisfied the comparison filtering criteria

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Microarray, Infection, Comparison, Binding Assay, Coagulation

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: C. pneumoniae stimulates TLR2 expression in rVSMCs. (A) Validation of TLR2 gene expression profiling using quantitative real-time RT-PCR normalized to GAPDH expression. Data shown are mean values of PCR replicates from individual groups. (B) TLR2 mRNA expression at the indicated time points after C. pneumoniae infection. rVSMCs infected with C. pneumoniae (5 × 105 IFU) for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h or control cells were lysed to prepare the total RNA. cDNA was amplified for 30 cycles, and PCR products were separated by agarose gel electrophoresis. Relative TLR2 mRNA expression levels determined by standard PCR (n = 3 replicates per group). (C) C. pneumoniae infection stimulates TLR2 protein expression in rVSMCs. Cells were infected with C. pneumoniae for 0 h, 12 h, or 24 h. Cell lysates were separated by SDS-PAGE, and blots were probed with anti-TLR2 and anti-β-actin antibodies, followed by donkey anti-goat IgG-HRP and goat anti-mouse IgG-HRP antibodies, and developed with enhanced chemiluminescence (ECL).

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Infection, Control, Amplification, Agarose Gel Electrophoresis, SDS Page

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Confocal microscopy analysis of the TLR2 expression pattern in C. pneumoniae-infected rVSMCs. rVSMCs were grown in coverslips and infected with C. pneumoniae for 60 h and then were fixed, permeabilized, and stained using specific antibodies. C. pneumoniae inclusions were observed using a mouse polyclonal antibody to C. pneumoniae. Fluorescence micrographs were stained with TLR2-specific antibody. Cell nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole).

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Confocal Microscopy, Expressing, Infection, Staining, Fluorescence

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Effects of TLR2 on rVSMC migration induced by C. pneumoniae infection. The TLR2-neutralizing antibody was added 1 h before C. pneumoniae infection. (A) Wound healing assay. “Scratch wounds” were created by scraping the confluent cell monolayer with a sterile pipette tip, and then cells were infected with C. pneumoniae at an infectious dose of 5 × 105 IFU. Photographs were taken of the same wounded area of each well at 0 h and 24 h. The scratched regions were photographed under an inverted Nikon microscope (×100 magnification) at 24 h after C. pneumoniae infection. Migration velocity is presented as a ratio of the cellular recoverage area to the whole wound area. *, P < 0.05 versus control; **, P < 0.05 versus C. pneumoniae infection group. (B) Transwell migration assay. Cell morphology was observed by staining with 0.1% crystallin violet. The number of cells that had migrated through the pores was quantified by counting nine independent visual fields using a microscope (×200 magnification). *, P < 0.05 versus control; **, P < 0.05 versus the C. pneumoniae infection group.

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Migration, Infection, Wound Healing Assay, Sterility, Transferring, Microscopy, Control, Transwell Migration Assay, Staining

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: The TLR2-neutralizing antibody suppresses Akt phosphorylation induced by C. pneumoniae infection. rVSMCs cultured for 24 h in 6-well plates were incubated with the TLR2-neutralizing antibody (10 μg/ml) and then infected with C. pneumoniae. Equal amounts of protein lysates were subjected to SDS-PAGE, and blots were probed with anti-Akt, anti-phospho-Akt (Ser 473), and anti-β-actin antibodies, followed by the corresponding horseradish peroxidase-conjugated secondary antibodies, and developed with ECL. P-Akt indicates phosphorylated Akt. *, P < 0.05 versus control; **, P < 0.05 versus the C. pneumoniae infection group.

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Phospho-proteomics, Infection, Cell Culture, Incubation, SDS Page, Control

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a , c ) Lung sections evaluated by H&E staining of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice at the age of ( a ) 3 and ( c ) 6 months. ( b ) Immunofluorescence for Mac-2 and p-S6 in lung sections of 3 month-old mice. Boxed region shows p-S6 staining only. ( d ) Image of a Tsc2 fl/fl Lyz2 -Cre mouse at the age of 6 months (left) and immunohistochemistry of Mac-2 of a paw (right). ( e ) Immunofluorescence for Mac-2 and F4/80 in paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice. ( f ) Flow cytometric scatterplot of the G1 population of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre peritoneal macrophages. ( g ) Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre bone marrow was injected into irradiated wild-type recipient mice and evaluated after 3 months by histology. Left panel: H&E staining of lung sections. Right panel: IHC of Mac-2 in liver. ( h ) Flow cytometry analysis of hypertrophic macrophages in the lungs of Tsc2 fl/fl (n=5) and Tsc2 fl/fl Lyz2 -Cre (n= 4) mice. Shown are means. ( i ) Expression of the indicated mRNAs in the lungs of 6 month-old mice. Shown are means ± SE (n= 5). *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant (Student's t test). Representative histological images or plots are from one mouse; each analysis was performed on three ( b , d , e , g ) or five ( a , c,f ) mice per genotype. Numerical data are representative of two independent ( h ) or cumulative of two independent ( i ) experiments. Scale bars, 100 μm ( a , c , e , g ), 10 μm ( b ), 1 mm ( d ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining, Immunofluorescence, Immunohistochemistry, Injection, Irradiation, Flow Cytometry, Expressing

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Immunoblots of CSF1-deprived Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre bone marrow-derived macrophages (BMDM), treated for 18 hours with 100 nM rapamycin or solvent control, hybridized with the indicated antibodies. ( b ) Surface expression of the indicated proteins on Tsc2 fl/fl (n=6) and Tsc2 fl/fl Lyz2 -Cre (n=4) BMDM after differentiation. ( c ) Left: Images of IL-4 stimulated (10 ng/ml) Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM treated with solvent or 100 nM rapamycin for four days. Right: Quantification of cluster formation. Clusters in three pictures per condition (4x magnification) were counted (n= 3). ( d ) Flow cytometry scatterplot of CSF1-deprived BMDM on day 7. ( e,f ) Cell cycle analysis of ( e ) BMDM by 7-AAD and EdU staining and of ( f ) peritoneal macrophages by PI staining from Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice (n= 5). ( g ) IHC of Ki-67 in lung sections of 3 month-old Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice. ( h ) Proliferation analysis of BMDM stimulated with 40 ng/ml CSF1 and treated with solvent or 100 nM rapamycin (n=4). Shown are means ± SE ( b , f,h ) or SD ( c ). *p < 0.001 (Student's t test) ( c , f ). Data are representative of three ( a , d , g,c ) or two ( e ) or cumulative from two independent experiments ( b,f,h ) Scale bar, 100 µm ( c ), 50 μm ( g ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Western Blot, Derivative Assay, Solvent, Control, Expressing, Flow Cytometry, Cell Cycle Assay, Staining

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Scatterplot of global gene-expression profiles of CSF1-deprived Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM. Blue dots, 426 genes significantly higher expressed in Tsc2 fl/fl BMDM; red dots, 401 genes significantly higher expressed in Tsc2 fl/fl Lyz2 -Cre BMDM. ( b ) Gene-set enrichment analysis (GSEA) of hallmark gene sets (H.all) from the Molecular Signatures Database of the Broad Institute, showing the most significantly enriched gene sets in Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM and their normalized enrichment scores (NES) as well as their false discovery rates (FDR). ( c,d ) GSEA plot of the E2F targets (c) and Tnfa signaling via NFKB (d) gene signature in Tsc2 fl/fl Lyz2 -Cre BMDM relative to Tsc2 fl/fl BMDM from the analysis in (b) with members of the gene set presented in the ranked list of genes ('bar code' below) and the signal-to-noise ranking metric (bar at bottom). ( e ) GSEA of the 'KEGG_apoptosis' gene signature in Tsc2 fl/fl Lyz2 -Cre BMDM relative to Tsc2 fl/fl BMDM. ( f ) The most significantly enriched transcription factor target gene sets (C3.TFT) from the Molecular Signatures Database in unstimulated Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM derived by GSEA. NES, normalized enrichment score; FDR, false discovery rate. Data are from one experiment with four biological replicates per genotype obtained from two independent experiments.

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Gene Expression, Derivative Assay

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a,b,c ) BMDM were treated with 100 nM rapamycin, solvent control and 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. ( d ) IHC of CDK4 in lung sections of 3 month-old mice. ( e ) Left, analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of the CKD4 inhibitor PD-0332991 (n= 4). Right, cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 µM PD-0332991 for 18 h (n= 3). ( f,g ) Tsc2 fl/fl Lyz2 -Cre bone marrow was injected into irradiated wild-type recipient mice. After ten days mice were treated daily with PD-0332991 (n=10) or solvent control (n=10) daily for two month. ( f ) Lung (left panel) and liver (right panel) sections were evaluated by IHC of Mac-2. ( g ) Area of Mac-2 positive cells compared to total lung area of the treated mice. Two random images per animal were evaluated. ( h ) BMDM were treated with 1 µM PD-0332991 or solvent control and stimulated with 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SE ( e ) or SD ( g ). *p < 0.01, **p < 0.001 (Student's t test). Data are representative of three ( a , b , c , d ), one ( f,g ) or two ( h ) independent, or cumulative of two ( e ) experiments. Scale bar, 40 μm ( d ), 100 μm ( f ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Solvent, Control, Western Blot, Cell Cycle Assay, Injection, Irradiation

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMDM reported in units of mpH/min and pmol/min, respectively (n=8). ( b ) Uptake of 2-NBDG in BMDM treated with solvent control or 100 nM rapamycin for 18 h; analyzed by flow cytometry (n=4). ( c ) Uptake of 2-NBDG in peritoneal macrophages from Tsc2 fl/fl (n=4) and Tsc2 fl/fl Lyz2 -Cre (n=5) mice. ( d ) Total amount of glucose in lung tissue of mice (n=10). ( e ) Mitotracker Green staining analyzed by flow cytometry of BMDM treated with solvent control or 100 nM rapamycin for 18 h (n=4). ( f ) Immunofluorescence for F-ATPase β of BMDM deprived of CSF1. ( g ) ECAR of Tsc2 fl/fl Lyz2 -Cre BMDM that were treated for 30 min with solvent or 1 µM PD-0332991 (n=8). ( h ) ECAR of Tsc2 fl/fl that were treated overnight with solvent or 10 ng/ml CSF1 and at the indicated time point with 1 µM PD-0332991 (n=8). ( i ) Images of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity on frozen lung sections of mice in situ . Sections were additionally stained for Mac-2, p-S6, and DAPI. ( j ) Fraction (in %) of p-S6-positive Mac-2 macrophages in lungs of mice with high activities of GAPDH, lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and succinate dehydrogenase (SDH) (n=3-4). ( k ) Analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of 2-DG (n= 4). ( l ) Cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 mM 2-DG for 18 h (n= 3). ( m ) BMDM were treated with 1 mM 2-DG or solvent control and then stimulated with 10 ng/ml CSF1 for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SD ( a , g , h , j ) or SE ( b , c , e , k , l ) or boxplots with means, 25 th to 75 th percentile and minimum to maximum bars ( d ). *p<0.05, **p<0.001, ***p<0.001 (Student's t test). Data are representative of one ( d ) or two ( a,f-j,m ) independent experiments or cumulative of 2 experiments ( b,c,e,k,l) . Scale bar, 10 μm ( f ), 100 μm ( i ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Solvent, Control, Flow Cytometry, Staining, Immunofluorescence, Activity Assay, In Situ, Cell Cycle Assay, Western Blot

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Sections of three sarcoidosis patients stained with p-S6 by IHC. ( b ) Immunofluorescence for Mac-2 and p-S6 in a sarcoidosis granuloma. ( c ) GSEA analysis of the ‘mTORC1 signaling’, ‘E2F targets’, and ´Glycolysis´ gene signatures in progressive relative to self-limiting sarcoidosis from the data of Lockstone et al . ( d ) An unsupervised cluster analysis of the microarray data of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM performed with the genes that were differentially expressed in progressive relative to self-limiting sarcoidosis patients. ( e ) IHC for p-S6 and Ki-67 of granulomas from sarcoidosis patients. ( f ) Immunofluorescence for Mac-2 and Ki-67 in a sarcoidosis granuloma. ( g ) Relationship between p-S6 and Ki-67 expression in granulomas of 27 human sarcoidosis biopsies. Ki-67 high, > 5 % Ki-67 positive cells in the granuloma; Ki-67 low, < 5 % Ki-67 positive cells in the granulomas. The relationship was investigated using fisher’s exact test. Data are representative of 27 human sarcoidosis patients ( a , b , e , f ) or from 8 self-limiting and 7 progressive sarcoidosis patients ( c , d ). Scale bar, 200 μm ( a ), 20 μm ( b ), 100 μm ( e,f ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining, Immunofluorescence, Microarray, Expressing

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) 2 month-old and ( b ) 6 month-old Tsc2 fl/fl Lyz2 -Cre mice were treated with placebo or everolimus for three weeks and lung sections were evaluated by H&E staining. ( c ) Paw and tail images of a 6 month-old Tsc2 fl/fl Lyz2 -Cre mouse at day 0 and 14 of everolimus treatment. ( d ) IHC for Mac-2 of paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice treated with placebo or everolimus. ( e ) IHC for p-S6 and cleaved caspase 3 of paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice treated with placebo or everolimus for two days. Representative histological images are from one mouse; each analysis was performed on four ( a , b , c ) or three ( d , e ) mice per genotype. Scale bar, 40 μm ( a ), 100 μm ( b ), 1 mm ( d ), 50 μm ( e ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining