Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 1. PDL1 expression in cancer and noncancer cell lines. A, relative expression of PDL1 from total RNA isolated from cell lines. B, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by IHC. C, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by flow cytometry.

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Isolation, Cytometry

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 2. Relative expression of PDL1 in 38 osteosarcoma specimens. PDL1 expression was evaluated from total RNA by quantitative real-time RT-PCR and showed that the expression levels range over 4 log (5,000-fold).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 3. Overall survival of 37 patients with osteosarcoma in relation to PDL1 gene expression. The median overall survival for PDL1-low patients was 89 months compared with 28 months for PDL1-high patients, which showed a trend but was not statistically significant (P ¼ 0.0544).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 4. Correlation between PDL1 gene expression and TILs by IHC. A, representative TILs in osteosarcoma tissues (400); score 0, no TILs; 1, rare/few TILs; 2, brisk/prominent TILs. B, significant positive correlation was shown between PDL1 gene expression and TILs in patients with osteosarcoma (P ¼ 0.0117).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 5. Characterization of the origins of metastases. A, PDL1 expression is significantly higher in metastatic osteosarcoma tumors that originate from lung than from other locations (P ¼ 0.0024). B, TILs also exhibit a positive correlation with pulmonary osteosarcoma metastasis compared with nonpulmonary metastasis (P ¼ 0.0443).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Schematic representation of luciferase screening approach. Upper panel shows the four target predictions software used for in silico analysis. Bottom panel indicates the main steps performed in the high‐throughput screening. B Upper panel, sequence interaction of miR‐182‐3p with the target site of the wild type 3′UTR of TRF2 in human. Bottom panel, generation of mutant 3′UTR of TRF2 luciferase construct containing the deletion of target site for miR‐182‐3p. C–E Luciferase reporter assay in HeLa cells using the synthetic miR‐Control or miR‐182‐3p in combination with the wild type (C) or the mutant 3′UTR of TRF2 construct (D) or the wild type 3′UTR of TRF1 (E). F, G Western blotting for TRF2 expression in telomerase‐positive (HeLa, HCT116, MDA‐MB‐231, MDA‐MB‐436) and ALT‐positive (U2‐OS, Saos‐2) cells transiently transfected with miR‐Control or miR‐182‐3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. H U2‐OS cells transiently transfected with the miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor were assayed by quantitative immunofluorescence for TRF2 3 days post‐transfection. Left panel, representative images. Scale bar: 10 μm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N = number of analyzed nuclei. Red bar indicates mean value. I U2‐OS cells transfected as described in (H) were assayed by immunofluorescence combined with telomeric FISH. Left panel, representative images of co‐localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10 μm. Right panel, co‐localizations were analyzed using ImageJ software. N = number of analyzed nuclei. Data information: For (C–G and I), data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by Student's t ‐test; for (H), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Luciferase, Software, In Silico, High Throughput Screening Assay, Sequencing, Mutagenesis, Construct, Reporter Assay, Western Blot, Expressing, Transfection, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Results of high‐throughput luciferase screening performed in Hela cells using the wild type 3′UTR‐TRF2 vector in combination with each of the 54 miRNAs selected by in silico analysis. Three days post‐transfection, luciferase ratio (Renilla:Firefly) of each miRNA was calculated, the control miRNA was set “1.” Renilla:Firefly ratios < 1 indicate target specificity of candidate miRNAs for the 3′UTR of TRF2. miRNAs near to the ratio of 0.5 were considered for further analysis. Two biological replicates were performed. B HeLa cells transiently transfected with the indicated miRNAs (miR‐Control, miR‐182‐3p, miR‐519e‐5p, miR‐296‐3p) were assayed by western blotting. Upper panel, quantification of TRF2 expression. Bottom panel, representative images of TRF2, TRF1 and RAP1 are shown, actin was used as loading control. C Analysis of TRF2 mRNA expression performed by qPCR in four different cancer cell lines (HeLa, MDA‐MB‐231, MDA‐MB‐436, U2‐OS) 3 days post‐transfection with miR‐Control or miR‐182‐3p. The control miRNA was set “1.” Three independent experiments were performed. D, E Telomeric ChIP assay in MDA‐MB‐231 (D) and U2‐OS cells (E). Quantification of TRF2 enrichment at telomeric repeats, in the different conditions, is shown in the table under the respective figure. Alu probe and Rabbit IgG were used as negative control for the assay. Data information: For (A), data are presented as mean values. For (B, C), data are presented as mean values ± SD and Student t‐ test was used to calculate statistical significance. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: High Throughput Screening Assay, Luciferase, Plasmid Preparation, In Silico, Transfection, Western Blot, Expressing, Negative Control

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A MDA‐MB‐231 cells were transiently transfected with the indicated miRNAs or siRNA. The indicated DNA damage markers were assayed by western blotting. Actin was used as loading control. B Telomeric DNA FISH performed in MDA‐MB‐231 transiently transfected with the indicated miRNAs. Telomere length was measured by TLF software and indicated as arbitrary fluorescence unit (a.f.u). N = number of analyzed nuclei. Black bar indicates mean value. C DNA damage markers were assayed by western blotting in HeLa cells. Actin was used as loading control. D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. Co‐localizations of γH2AX with telomeres are indicated as mean number of TIFs per nucleus. E Representative images and enlargements of co‐localizations of experiment described in D. F Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. G Representative images of co‐localizations relative to the experiment described in (F). H, I MDA‐MB‐231 and HeLa cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with miR‐Control or miR‐182‐3p. TRF2, pATM and γH2AX expression were assayed by western blotting. Actin was used as loading control. Data information: For (D) and (F), data are presented as mean values ± SD. Three independent replicates were performed. Scale bar: 10 μm. At least 60 nuclei were analyzed in (D) and (F). A Student t‐ test was used to calculate statistical significance. For (B), P values are determined by Mann–Whitney t ‐test. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Western Blot, Software, Fluorescence, Immunofluorescence, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analyzed. Representative images and enlargements of co‐localizations (white arrowheads) relative to the experiment described in (A). Scale bar: 10 μm. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images of co‐localizations (white arrowheads) relative to the experiment described in (C). Scale bar: 10 μm. Quantification of TIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. Representative images and enlargements relative to the experiment described in (E). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Quantification of PIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images relative to the experiment described in (G). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Data information: For (A, C, E, G) data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. At least 60 nuclei were analyzed for each experimental condition. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐436 and MDA‐MB‐231 cells underwent two rounds of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24 h up to a maximum of 3 days. The percentage of cell confluence was analyzed. C, D Cell number of MDA‐MB‐436 (C) and MDA‐MB‐231 (D) cells and TRF2 expression were analyzed by automatic cell count and by western blotting at the end of the experiment described in (A) and (B). Actin was used as loading control. E Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐436 at the end of the second cycle of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Red boxes indicate early and late apoptotic cells. F Quantification of Annexin V‐positive cells (%) of experiment described in (E). G Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐231 as described in (E). H Quantification of Annexin V‐positive cells (%) of experiment described in (G). I, J MDA‐MB‐436 cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72 h post‐transfection. Data information: For (A, B) data are shown as mean ± SEM. For (C, D, F, H, I, J), data are shown as mean ± SD. For (A–D) and (I), three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. For (F), (H) and (J), two different biological replicates were performed. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Cell Counting, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Western blotting for TRF2 expression in BJ cells transiently transfected with miR‐Control or miR‐182‐3p. The graph represents the quantification of three independent experiments. Representative images are shown, Actin was used as loading control. Unspecific bands are indicated with (*). B, C Mean of γH2AX foci per nucleus was analyzed in BJ cells 72 h post‐transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in (C). D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72 h post‐transfection with the indicated miRNAs. Left panel: The mean number of TIFs per nucleus was analyzed. Right panel: Representative images and enlargements of co‐localizations. E Cell number of BJ cells was analyzed by automatic cell count at the end of the second round of transfection with miR‐Control or miR‐182‐3p. F FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in (E). G β‐Galactosidase assay in BJ cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. H–J IL‐6 (H), CXCL1 (I), IL‐8 (J) factors were analyzed by ELISA to evaluate the senescence‐associated secretory phenotype (SASP) in BJ cells treated as indicated in (G). Data information: For (A, B, D, E and G–J), a student t‐ test was used to calculate statistical significance. Scale bars (10 μm). P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Cell Counting, Staining, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A TRF2 and γH2AX expression after two rounds of transfection with the indicated miRNAs, was analyzed by western blotting in MCF10A cells. Actin was used as loading control. B–E The mean number of γH2AX foci (B) and TIFs (D) per nucleus were analyzed 72 h post‐transfection with the indicated mimic miRNAs in MCF10A cells. Representative images (C) and (E) are referred to the experiment showed in (B) and (D) respectively. F, G Cell confluence (F) of MCF10A was monitored by Incucyte, every 24 h starting from the day of the second transfection, and cell number (G) was counted at the end of experiment (day 4). H–I Cell cycle progression analysis by PI staining (H) and cell death analysis by Annexin V assay (I) were performed in MCF10A upon two rounds of transfection with the indicated miRNAs. J β‐Galactosidase assay in MCF10A cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. Data information: Panels (B, D, F, G, J) data are presented as mean values ± SD. A Student t‐ test was used to calculate statistical significance. P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Transfection, Western Blot, Staining, Annexin V Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐231 (A) and MDA‐MB‐436 (B) tumor xenografts were treated with LNPs‐empty, LNPs‐miR‐Control or by LNPs‐miR‐182‐3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20 μg of LNPs‐miR‐Control, LNPs‐miR‐182‐3p or equivalent volume of LNPs‐empty as indicated in the scheduling. The mean of tumor volumes ( n = 5 per group) is shown. C, D Tumors from mice treated in (A) and (B) were processed to measure miR‐182‐3p expression by TaqMan qPCR. E Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA‐MB‐231 human breast cancer xenografts. Scale bar: 50 μm. F The histograms show the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, TUNEL or CD31 staining. The analyses were performed on three mice per group, and the points represent the number of field analyzed for each condition. G, H Luminescent MDA‐MB‐436 cells were injected into the brain and monitored by IVIS imaging system. After 1 week from implant, treatment with LNPs‐miR‐Control and LNPs‐miR‐182‐3p was performed as indicated in (A) and (B). Representative images from in vivo (upper panel) or ex‐vivo (bottom panel) brain tumors are shown in (G). Boxplots (H) show the measurement of photons for each brain tumor ( n = 5 per group) acquired at the indicated times. Data information: For (A, B, F), data are shown as mean ± SD. For (C, D, H), the line in the middle of the box plot denotes a median value, the limits of box represent the interquartile range (25 th to 75 th percentiles), while, the whiskers denote the minimum to maximum values. For (A–D) and (H), P values are determined by unpaired two‐tailed t‐ test; for (F), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, TUNEL Assay, Staining, Injection, Imaging, In Vivo, Ex Vivo, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: The organs (brain, liver, kidney) taken from mice, previously engrafted with MDA‐MB‐231 cells and treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p, were assayed for miR‐182‐3p expression by TaqMan qPCR. Representative images show IHC analysis on tumor samples, from mice bearing MDA‐MB‐436 human breast cancer xenografts, with the indicated markers. Scale bar: 50 μm. The histograms show the expression of TRF2 indicated as immunoreactivity score (IRS) and the percentage of positive cells to γH2AX, TIUNEL or CD31 staining in MDA‐MB‐436 xenografts. Three mice per group were analyzed, the points represent the number of field analyzed for each condition. Data information: For (A, C), data are presented as mean values ± SD. Statistical significance using unpaired (A) or Mann–Whitney t‐ test (C) was calculated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B PDTCs #1 and #2 underwent two rounds of transfection with miR‐Control or miR‐182‐3p. Three days after the second transfection, miR‐182‐3p and TRF2 expression were analyzed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. C, D Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50 μm. At least 85 3D cells were analyzed for each experimental condition. E NSG mice implanted with breast PDTX (#2) were treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p as indicated in the scheduling. Caliper measurement of tumors was taken at the indicated days. The mean of tumor volumes ( n = 5 per group) is shown. F miR‐182‐3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. G Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in (E). Scale bar: 50 μm. H The histograms show the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and TUNEL. The analysis was performed on three mice per group, the points represent the number of field analyzed for each condition. Data information: For (A–F) and (H), data are shown as mean ± SD. For (A–F), P values are determined by unpaired two‐tailed t‐ test; for (H), P values are determined by Mann–Whitney t ‐test. For the experiments showed in (A, B) and (C, D) two or three biological replicates were performed, respectively. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Western Blot, TUNEL Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Representative images of intestine sections from mice previously treated with LNPs‐Empty or LNPs‐miR‐182‐3p. H&E staining (scale bar: 200 μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50 μm). B, C Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX‐positive cells (%) (C) on intestine samples. D Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of skin samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). E, F Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX‐positive cells (%) (F) on skin samples. G Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). H, I Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX‐positive cells (%) (I) on bone marrow samples. Data information: For (B, C, E, F, H, I), data are shown as mean ± SD. A Mann–Whitney test t‐ test was used to calculate statistical significance. Four mice per group were analyzed, the points represent the number of field analyzed for each condition. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: British Journal of Pharmacology

Article Title: NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

doi: 10.1111/bph.12049

Figure Lengend Snippet: Primers used for the RT-PCR experiments

Article Snippet: Cells were washed two times with PBS, blocked and permeabilized in PBS containing 10% normal donkey serum and 0.1% Triton X-100 for 30 min. Once again, cells were washed with PBS and incubated with primary antibodies: anti-K Ca 2.1 (KCNN1, SK1); anti-K Ca 2.2 (KCNN2, SK2); anti-K Ca 2.3 (KCNN3, SK3) or anti-K Ca 3.1 (KCNN4, IK, SK4); 1:100; Alomone Labs (Jerusalem, Israel) at 37°C for 1 h. Next, cells were washed two times with PBS and labelled with secondary antibodies (Cy3-conjugated anti-rabbit IgG, at 1:100, PBS/3% normal donkey serum/0.01% Triton X-100; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h in the dark.

Techniques:

Journal: British Journal of Pharmacology

Article Title: NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

doi: 10.1111/bph.12049

Figure Lengend Snippet: RT-PCR detection of mRNA messages for SK3 channels in DSM whole tissue and freshly isolated single cells. mRNA expression for the SK3 channel was detected in DSM whole tissue and freshly isolated DSM single cells, whereas no mRNA expression was observed for SK1, SK2 and IK (SK4) channels in DSM single cells. Rat brain was used as a positive control. Negative control experiments carried out with omission of RT-enzyme (−RT) in reaction mixtures showed no products. Illustrated gel images are representations of at least four independent RT-PCR experiments based on RNA extracted from four rats.

Article Snippet: Cells were washed two times with PBS, blocked and permeabilized in PBS containing 10% normal donkey serum and 0.1% Triton X-100 for 30 min. Once again, cells were washed with PBS and incubated with primary antibodies: anti-K Ca 2.1 (KCNN1, SK1); anti-K Ca 2.2 (KCNN2, SK2); anti-K Ca 2.3 (KCNN3, SK3) or anti-K Ca 3.1 (KCNN4, IK, SK4); 1:100; Alomone Labs (Jerusalem, Israel) at 37°C for 1 h. Next, cells were washed two times with PBS and labelled with secondary antibodies (Cy3-conjugated anti-rabbit IgG, at 1:100, PBS/3% normal donkey serum/0.01% Triton X-100; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h in the dark.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Positive Control, Negative Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: TREK2 channel expression in human bladder cancer cells. (A) Messenger RNA (mRNA) of ion channels related to the TREK1 (Accession No; AF129399) and TREK2 (Accession No; AF279890) were amplified by reverse transcription-polymerase chain reaction (RT-PCR) analysis. TREK1 (355base pair [bp]) and TREK2 (291 bp) were detected. (B) Immunoblot showed presence of TREK2 channel protein in the human bladder cancer 253J cell line. TREK2 transfected CHO cells were used as a control. (C) Representative confocal microscopic analysis of TREK2 in bladder cancer cell line 253J. Cells were stained with an anti-TREK2 antibody and F-actin. Scale bar, 20 µm.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Staining

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: The physiologic properties of TREK2 at a single channel level in bladder cancer 253J cells. (A) TREK2 in CHO cells transfected with DNA encoding TREK2 and GFP and in 253J cells was measured in the excised inside-out patch configuration at the holding potential values shown on the left. The current trace was obtained in symmetrical 150 mM KCl solutions. The letters "c" and "o" represent the "closed" and "open" states of the channels, respectively. (B) The I-V relationships showed inward rectification, and each point is the mean of 4 experiments with standard error (S.E) represented by the error bars. (C~E) Current tracing showed arachidonic acid, intracellular pH, and mechanosensitivity of the native TREK2-like channel in 253J cells at -60 mV, +40 mV, and -40 mV. Negative pressure (-10 mmHg or -20 mmHg) was applied through the pipette. The panel below each of the figures shows the single channel trace on an expanded time scale.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Transfection, Transferring

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: The effect of TREK2 knockdown on the growth of 253J bladder cancer cells. (A and B) TREK2 mRNA and protein levels in 253J cell were determined after knockdown of TREK2 by siRNA through qRT-PCR and Western blot. Expression of TREK2 mRNA after transfection of TREK2 siRNA or negative control siRNA was normalized. TREK2 protein was examined by Western blot analysis after transfection of the negative control or TREK2 siRNA in 253J cells. GAPDH was used as a control. (C) The membrane potential was measured at current clamp (I=0) in a whole cell patch configuration. The 253J cells were treated with FITC-labeled negative control siRNA and FITC-labeled TREK2 siRNA for 72 hours. The data were represented as the mean±S.E. (t-test, p value <0.05). (D) The antiproliferative effect of TREK2 knockdown by siRNA in 253J cells. Cells were treated for three days with TREK2 siRNA or (-)control siRNA in 2% serum culture media. After treatment, proliferation was measured by XTT assay. Error bars represent the mean±S.E for 38 separate experiments. Asterisks indicate values that are different from the respective control (t-test, p<0.05). (E) Effect of TREK2 siRNA on 253J cell growth. Cells were captured 48 hours after transfection with TREK2 siRNA using a Nikon microscope at 10×10 magnification. Scale bar, 100 µm.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Negative Control, Labeling, XTT Assay, Microscopy

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: Cell cycle arrest at G0/G1 after TREK2 siRNA transfection in bladder cancer 253J cells. (A) The bar graph shows that TREK2 siRNA treated cells resulted in an increased percentage of cells in G0/G1 and a decreased percentage of cells in the S phase of the cell cycle (t-test p<0.05) (B) Representative Western blot showing changes in the levels of associated proteins in cell cycle arrest of human bladder cancer 253J cells. Knockdown of TREK2 decreased the expression of cyclin D1, cyclin D3, cdk2, cdk4, and cdk6 and increased protein levels of p21 and p53.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Transfection, Western Blot, Expressing

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor expression in synovial tissue of patients with rheumatoid arthritis. The representative results of HE staining (Figure 2A), immunofluorescence anti-CTGF antibody staining (Figure 2B; green), and anti-F4/80 antibody staining (Figure 2C; red) are shown using surgical samples from RA and OA patients. The samples were counterstained by DAPI (blue) for nuclear staining and merge images are shown (Figure 2D). A strong expression of CTGF and F4/80 was observed in the samples of RA compared to OA and the CTGF expression cells were not overlapped with F/40 expression cells indicating that CTGF is upregulated in synovial fibroblasts rather than macrophages.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Expressing, Staining, Immunofluorescence

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Synergistic effects of connective tissue growth factor on M-CSF/sRANKL-mediated osteoclastic function. Figure 5A shows the results of the resorption of osteoclasts on calcium phosphate. Vacant regions indicated by arrows represent the areas where the osteoclasts actually absorbed. There was no vacant region in negative control cells (M-CSF alone). In contrast to negative control, significant vacant regions were observed in osteoclasts induced by M-CSF/sRANKL. CTGF further expanded the vacant areas in combination with M-CSF/sRANKL and anti-CTGF antibody neutralized this effect. Figure 5B shows the levels of expression of osteoclasts specific proteases (MMP-9 and cathepsin-K) measured by quantitative real time RT-PCR. Synergistic effect of CTGF was also observed for M-CSF/sRANKL-mediated osteoclastogenesis. Bars in Figure 5B indicate the SD.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Negative Control, Expressing, Quantitative RT-PCR

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor mediate ERK1/2 and focal adhesion kinase activation through integrin αVβ3 signal transduction. Figure 6A shows the immnoprecipitation and immunoblotting analysis. The cell extracts of osteoclasts stimulated with recombinant CTGF (10 or 50 ng/ml) at 60 minutes were precipitated using anti-integrin αVβ3 antibody and subsequently blotted with anti- phosphorylated ERK1/ERK2, conventional ERK1/ERK2, and integrin αVβ3 antibodies respectively. The phosphorylated ERK1/ERK2 was recruited with integrin αVβ3 by CTGF stimulation. Figure 6B shows the immunoblotting analysis using anti- phosphorylated FAK, conventional FAK, and β-actin antibodies in the osteoclasts extracts treated with CTGF (10 ng/ml) at 5, 15, 60, and 120 minutes in the presence or absence of anti-CTGF antibody (1 μg/ml). CTGF stimulation resulted in phosphorylation of FAK from 60 minutes and this effect was neutralized by anti-CTGF antibody suggesting activation of signal transduction pathways through integrin αVβ3.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Activation Assay, Transduction, Western Blot, Recombinant, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) 1×10 6 SL-1 and SL-3 cells were cultured for 24 hr, whole-cell lysate (WCL) and supernatant (SUP) were prepared and subjected to cytokine array analyses. Each cytokine has one pair of duplicate spots. A1-2, A23-24, and F1-2 are experimental positive control, and F23-24 is an experimental negative control. (B) C57BL/6 mice were intranasally inoculated with 5×10 4 PFU of MHV68-H2bYFP or mock inoculated with PBS. At day 16 post-infection, splenocytes were isolated and subjected to flow cytometry, the flow plot represented the strategy gating YFP+ MHV68 infected cells (left panel); serum was prepared from 10 virus-infected mice or mock-infected mice, followed by IL16 ELISA assay (right panel). Histograms represented mean ±SD of 10 individual mice (two experiments, n = 5 for each experiment). p value was determined by two-tailed unpaired t-test. (C) WT MEFs were infected with MHV68 at an MOI of 1, total RNA was isolated from infected cells harvested at the indicated time points and subjected to qRT-PCR analyses with specific primers corresponding to IL16 and MHV68 ORF50 gene. The relative RNA amount was normalized to GAPDH in each sample. Histograms represented the mean of three independent biological replicates ±SD, p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Cell Culture, Positive Control, Negative Control, Infection, Isolation, Flow Cytometry, Virus, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) Intracellular staining of IL16 in splenocytes isolated from IL16+/+, IL16+/-, and IL16-/- mice. (B) Immunoblot detection of IL16 expression in splenocytes isolated from IL16+/+, IL16+/-, and IL16-/- mice. (C) Representative flow plots showed flow cytometric analyses of splenocytes from IL16+/+ (WT) and IL16-/- (KO) mice. (D) The statistic analyses of CD4+ T, CD8+ T, and B cells (Left panel); follicular (FC) B, marginal zone (MZ) B, and mature B cells (Right panel) in splenocytes from WT and IL16 KO mice. Histograms represented mean ±SD of 8 individual mice (two experiments, n = 4 for each experiment). ns = not significant.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Staining, Isolation, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO MEFs were infected with MHV68 at an MOI of 1 or 0.05. The infected cells were harvested at the indicated time points and immunoblot analyses were performed with specific antibodies as indicated. Actin was used as a loading control. (B) WT and IL16 KO MEFs were infected with MHV68 at an MOI of 5 or 0.05. The supernatant was harvested at the indicated times and viral titers were determined by TCID50 assay. Results are means from triplicate samples. Error bars represented standard deviations. ns = not significant. (C) WT and IL16 KO mice were intranasally infected with 5×10 4 PFU of MHV68. Lungs of infected mice were collected at day 4 and 7 post-infection. Virus titers were determined by TCID50 assay. Data represented one of two independent experiments with 5 or 7 mice per group. ns = not significant. Each symbol represented an individual mouse. The horizon line indicated geometric mean titer. (D) Vector or IL16-expressing plasmids with Flag tag were transfected into BHK21 cells for 24 hr, followed by MHV68 infection at an MOI of 5 or 0.05. The infected cells were harvested at the indicated time points and immunoblot analyses were performed with specific antibodies as indicated.–and + represents the cells transfected with vector and IL16-expressing plasmids with Flag tag, respectively. (E) Supernatant was harvested at the indicated times and viral titers were determined by TCID50 assay. Results were means from triplicate samples. Error bars represented standard deviations. ns = not significant.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Western Blot, Control, TCID50 Assay, Virus, Plasmid Preparation, Expressing, FLAG-tag, Transfection

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and three IL16 KO single clones were stimulated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr, respectively. Immunoblot analyses were performed with the indicated antibodies. GAPDH was used as a loading control. (B) WT and IL16 KO cells (clone E6) were stimulated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the indicated antibodies. GAPDH was used as a loading control. MHV68 viral genome was determined by qPCR with the primers specific to the MHV68 ORF50 coding region. The relative copy of the MHV68 viral genome was normalized to GAPDH in each sample. (C) The mRNA expression of MHV68 viral gene ORF73, ORF50, ORF59, and ORF25 was determined by qRT-PCR. The relative RNA amount was normalized to GAPDH in each sample. Histograms represented the mean of three independent biological replicates ±SD, p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Clone Assay, Western Blot, Control, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) The diagram showed the potential cleavage sites of IL16. (B) 293T cells were transfected with IL16 or mutants with Flag tag. At 48 hr post-transfection, the supernatant was collected and subjected to IL16 ELISA assay; whole-cell lysates (WCL) and supernatant (SUP) were prepared and subjected to immunoblot analyses with the indicated antibodies. (C) IL16 KO SL-1 cells were transfected with vector (Vec), IL16-, or IL16(D516A)-expressing plasmid with Flag tag, followed by anti-mouse Ig(G+M) treatment for 48 hr. The whole-cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Transfection, FLAG-tag, Enzyme-linked Immunosorbent Assay, Western Blot, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were inoculated intranasally with 5×10 4 PFU of MHV68-H2bYFP. Mice inoculated with 5×10 4 PFU of WT MHV68 were used as a control to gate YFP+ cells. Splenocytes were isolated at day 16 and day 18 post-infection. (A) Representative flow plots showing the identification of MHV68-infected YFP+ cells. (B) Frequency of YFP+ cells at day 16 post-infection. Results were compiled from two independent experiments with 8–9 mice per group. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency of infected cells. ns = not significant. (C) Frequency of splenocytes capable of reactivating virus by ex-vivo assay at day 16 post-infection. Serial dilutions of splenocytes were plated on MEFs and the presence of reactivating virus was determined by the presence of cytopathic effect (CPE). Representative results were from two independent experiments with 8–9 mice per group. (D) Frequency of YFP+ cells at day 18 post-infection. Results were compiled from two independent experiments with 10–12 mice per group. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency of infected cells. ns = not significant. (E) Frequency of splenocytes capable of reactivating virus by ex-vivo assay at day 18 post-infection. Data were generated from two independent experiments, 5 to 6 mice per experiment per group.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Control, Isolation, Infection, Virus, Ex Vivo, Generated

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were intranasally inoculated with 5×10 4 PFU of MHV68-H2bYFP and splenocytes were harvested at day 16 post-infection. (A) Representative flow plots showing the identification of MHV68-infected YFP+ cells. (B) Representative flow plots of YFP+ germinal center B cells (CD19 + CD95 + GL-7 + YFP + ). (C) Quantitation of the percentage of YFP+ germinal center B cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (D) Representative flow plots of YFP+ plasma cells (CD3 - YFP + B220 low CD138 + ). (E) Quantitation of the percentage of YFP+ plasma cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (F) Quantitation of the percentage of total germinal center B cells (CD19 + CD95 + GL-7 + ). (G) Quantitation of the percentage of total plasma cells (CD3 - B220 low CD138 + ). ns = not significant. p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Quantitation Assay, Clinical Proteomics, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were inoculated intranasally with 5×10 4 PFU of MHV68-H2bYFP. Splenocytes were isolated at day 16 post-infection and subjected to flow cytometry analyses. (A) Representative flow plots of CD4+ and CD8+ T cells from infected WT and IL16 KO mice. (B) Quantitation of the percentage of CD4+ and CD8+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (C) Representative flow plots of IFN-γ+CD4+ T cells from infected WT and IL16 KO mice. (D) Quantitation of the percentage of IFN-γ+CD4+ and CD44+CD4+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (E) Quantitation of the percentage of IL4+CD4+ and IL2+CD4+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (F) Representative flow plots of IFN-γ+ CD8+ T cells from infected WT and IL16 KO mice. (G) Quantitation of the percentage of IFN-γ+CD8+ and TNF-α+CD8+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. ns = not significant. p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Isolation, Infection, Flow Cytometry, Quantitation Assay, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) The murine M12 B lymphoma cells were transfected with renilla reporter, a luciferase reporter (pGL2) driven by RTA proximal promoter (RTAp), together with IL16-expressing plasmid with Flag tag or vector alone (Vec). Luciferase activity was normalized to renilla activity and Luciferase value was reported as fold increase in luciferase activity over basal promoter activity. Each sample was done in triplicate (two independent experiments). IL16 expression was detected by immunoblot with a Flag antibody. (B) M12 cells were transfected with RTAp together with vector (Vec), IL16-, IL16(D506A)- or IL16(D516A)-expressing plasmid with Flag tag. At 24 hr post-transfection, transfected cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 12 hr. IL16, IL16 (D506A), and IL16 (D516A) expression was detected by immunoblot with Flag antibody. Luciferase value was reported as fold increase in luciferase activity over basal promoter activity. Each sample was done in triplicate (two independent experiments).

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, FLAG-tag, Activity Assay, Western Blot

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO SL-1 cells were treated with (+) or without (-) 20 μM JAKs inhibitor AG490 for 1 hr, followed by stimulation with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. GAPDH was used as a loading control. (B) WT and IL16 KO SL-1 cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. (C) Quantitation of phosphorylated STAT relative to total STAT based on immunoblot detection in C using the ImageJ image analysis software.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Western Blot, Control, Quantitation Assay, Software

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO SL-1 cells were pretreated with DMSO, 40 μM, or 80 μM STAT5 inhibitor (STAT5-I) for 1 hr, followed by stimulation with anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. (B) SL-1 cells were transfected with vector, STAT3-expressing plasmid, or STAT3C-expressing plasmid with Flag tag, followed by anti-mouse Ig(G+M) (5 μg/mL) treatment for 48 hr. Immunoblot analyses were performed with the indicated antibodies. (C) WT and IL16 KO SL-1 cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the specific antibodies as indicated. (D) Quantitation of phosphorylated STAT3(Y705) relative to total STAT3 and p21 relative to GAPDH based on immunoblot detection in C using the ImageJ image analysis software. (E) WT and IL16 KO SL-1 cells were treated with DMSO or Orthovanadate (50 μM) in the presence or absence of anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the specific antibodies as indicated.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, FLAG-tag, Quantitation Assay, Software

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: MHV68 infection induces IL16 production, which, in turn, increases STAT3(Y705) phosphorylation, subsequently reduces p21 expression, and inhibits MHV68 reactivation. Meanwhile , IL16 partially inhibits RTA promoter activity and STAT3(S727) phosphorylation, contributing to the inhibition of MHV68 reactivation. Ultimately, MHV68-induced IL16 helps to maintain MHV68 latency.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Phospho-proteomics, Expressing, Activity Assay, Inhibition

Journal: Molecular Medicine Reports

Article Title: Girdin regulates the migration and invasion of glioma cells via the PI3K-Akt signaling pathway

doi: 10.3892/mmr.2015.4049

Figure Lengend Snippet: Girdin silencing inhibits the expression and activity of MMP-2 and MMP-9. (A and B) Changes in the mRNA levels of MMP-2 and MMP-9 were measured using reverse transcription-quantitative polymerase chain reaction following transfection. The relative mRNA expression levels were calculated using the 2 −ΔΔCt method. (C and D) Following transfection, changes in the protein levels of MMP-2 and MMP-9 were detected using western blot analysis. (E and F) Following transfection, gelatin zymography was performed to detect changes in the activities of MMP-2 and MMP-9. Each experiment was repeated three times. The experimental results are presented as the mean ± standard deviation. ** P<0.01, compared with the NC group. shRNA, short hairpin RNA; NC, negative control.

Article Snippet: Following washing with Tris-buffered saline with 0.05% Tween-20 (TBST), the membranes were incubated with the following primary antibodies at 4°C overnight: Rabbit anti-human polyclonal antibody against girdin (1:500 diluted; cat. no. bs-5150R; Bioss, Beijing, China); rabbit anti-human polyclonal antibody against MMP-2 (1:1,000 diluted; cat. no. WL0657); rabbit anti-human polyclonal antibody against MMP-9 (1:1,000 diluted; cat. no. WL0884); rabbit anti-human polyclonal antibody against P85α (1:1,000 diluted; cat. no. WL0191); rabbit anti-human polyclonal antibody against P110α (1:1,000 diluted; cat. no. WL0339); rabbit anti-human polyclonal antibody against AKT (1:1,000 diluted; cat. no. WL0003); rabbit anti-human polyclonal antibody against p-AKT (1:1,000 diluted; cat. no. WLP001).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Zymography, Standard Deviation, shRNA, Negative Control

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Schematic diagram of the positions and orientations of syncytin-1, PEX1 and GATAD1 genes. Arrows show genes’ orientations. The patterned squares represent exons. Dark squares indicate the location of the CpG islands in GATAD1 gene. Light grey square represents the syncytin-1 5′ LTR region. The solid lines at the bottom show the positions of amplicons of real-time PCR. Note the opposite orientations of GATAD1 and syncytin-1 genes, which bring the 3 [prime] region of GATAD1 to a closer vicinity of syncytin-1 gene.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Information of PCR primers

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques:

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: PEX1 and GATAD1 mRNA levels in first-trimester (1N, n=8), third-trimester normal (3N, n=14) and third-trimester preeclamptic (3P, =7) placentas. Real-time PCR were performed as described in Materials and methods. Data were standardized by the results from β-actin internal control. The mRNA levels from third-trimester normal (3N) placentas were set as 1. The averages and standard errors of each group were presented. a PEX1 mRNA levels. No significant difference was found between 1N and 3P, or 3N and 3P groups. b GATAD1 mRNA levels. Significantly higher GATAD1 mRNA levels were observed in 3N than in 1N; A significant reduction of GATAD1 mRNA levels were found in 3P compared to 3N group. **p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Protein levels of GATAD1 in the first-trimester (1N1-1N6), third-trimester normal (3N1-3N6), and third-trimester preeclamptic (3P1-3P6) placentas. The sizes of GATAD1 and β-actin proteins are 29 kDa and 42 kDa, respectively. a Western blotting performed using GATAD1-specifc antibodies. b Results of densitometry analyses showing a similar trend of changes to that of mRNA levels: GATAD1 protein expression was higher in 3N than 1N, and lower in 3P than 3N. The GATAD1 expression data were standardized by the results from β-actin. The protein levels from third-trimester normal (3N) placentas were set as 1. The averages and standard errors from each group were presented. **p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Representative results of immunohistochemistry (40×10). The paraffin-embedded placental tissues were sliced into 4 μm sections. The sections were processed as describe under Materials and methods. As negative control (bottom right panel), a section of first-trimester normal placenta was processed with the same procedures except for the absence of primary antibodies. GATAD1 protein was stained brown color. The nuclei were stained blue with haematoxylin. GATAD1 protein localized mostly in the cytoplasm and membrane of syncytiotrophoblasts (ST), and to a less extent, the cytoplasm and membrane of cytotrophoblasts (CT). Higher level of GATAD1 expression was found in third-trimester (upper right) than first-trimester (upper left panel) placenta. Preeclamptic (bottom left) placentas expressed decreased levels of GATAD1 protein compared to normal placentas.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Negative Control, Staining, Expressing

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: GATAD1 gene methylation measured by COBRA. Following PCR amplification, DNA fragments representing the 5 [prime] and 3 [prime] regions of GATAD1 gene were digested with an excess of BstUI or TaqαI, respectively. Agarose gel electrophoresis was performed and DNA bands were visualized by ethidium bromide staining. a The absence of cleavage product (supposedly 147 bp and 107 bp) from 254 bp fragment indicated an largely unmethylated status of GATAD1 5 [prime] region in first-trimester (1N1 to 1N8), third-trimester normal (3N1 to 3N14) and Preeclamptic (3P1 to 3P7) placentas. b The 241 bp fragment representing the 3 [prime] region of GATAD1 was mostly cleaved, generating the 144 bp and 97 bp bands indicative of DNA methylation. c Densitometry analyses of the 3 [prime] methylation showing an increased methylation in 3N placentas compared to 1N, and decreased methylation levels in 3P placentas compared to 3N group. ** p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Methylation, Combined Bisulfite Restriction Analysis Assay, Amplification, Agarose Gel Electrophoresis, Staining, DNA Methylation Assay

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Bisulfite sequencing of the GATAD1 3 [prime] region. Bisulfite-converted DNA from 1N (n=5), 3N (n=5) and 3P (n=5) groups were PCR amplified, subcloned, and sequenced. a The typical sequencing result of the 3 [prime] region. Asterisks (*) mark CpG sites. The TaqαI recognition site used in COBRA is underlined. b GATAD1 3 [prime] bisulfate sequencing results. The solid and open circles represent the methylated and unmethylated cytosines, respectively, in CpGs dinucleotides contexts. The average methylation levels for each CpG site were presented in the bottom panels. c Quantitative comparison of the GATAD1 3 [prime] methylation among the three groups. 3N placentas displayed increased methylation levels compared to 1N, and 3P group exhibited decreased methylation levels compared to 3N. * p < 0.05; ** p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Methylation Sequencing, Amplification, Sequencing, Combined Bisulfite Restriction Analysis Assay, Methylation

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: The correlation between GATAD1 expression and GATAD1 3 [prime] methylation in human placentas (n=29). The Y-axis indicated GATAD1 mRNA levels and the X-axis represented GATAD1 3 [prime] methylation index. Spearman correlation analysis showed a highly significant positive correlation between GATAD1 mRNA levels and GATAD1 3 [prime] methylation levels among placental samples (r=0.62, p=0.0003).

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Expressing, Methylation

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Treatment with DNMT inhibitor led to a decreased GATAD1 3 [prime] DNA methylation and decreased GATAD1 expression. JAR cells were treated for 5 days with 0, 0.5, and 2.5 μM of 5-aza-deoxycytidine (ADC). a GATAD1 3 [prime] methylation was examined with COBRA. b Densitometry analyses indicated a dose-dependent decrease of GATAD1 3 [prime] DNA methylation following ADC treatment. c Results of real-time PCR showed a decrease of GATAD1 mRNA expression following ADC treatment. Data were standardized with the results from β-actin. Averages and standard errors were presented in the chart. **p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: DNA Methylation Assay, Expressing, Methylation, Combined Bisulfite Restriction Analysis Assay, Real-time Polymerase Chain Reaction