Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 1 Elevated expression of HER3 is frequently observed in TNBC clinical samples and cell lines and associates with poor clinical outcomes in TNBC patients. A, IHC assays were performed to examine HER3 protein expression in a tissue microarray (TMA) of TNBC specimens (n = 125). Representative im ages of negative (-), low (+), medium (++), and high (+++) staining intensity of HER3 expression were shown. The case number in each group was shown underneath. B, The heatmap showed HER3 mRNA expression in CCLE breast cancer cell lines (http://software.broadinstitute.org/morpheus). Red colors indicated TNBC cell lines. C, Analyses of overall survival (OS) or relapse-free survival (RFS) of basal-like breast cancer patients were performed using Kaplan- Meier Plotter (https://kmplot.com/analysis/). The expression levels of HER3 mRNA were determined in TCGA-curated basal-like breast cancer datasets and used for log rank tests to compare the survival curves of those with high (red) or low (black) HER3 expression

Article Snippet: Specificity of 4A7 was further determined by ELISA with human HER3 His Tag (ER3-H5223) protein (ACROBiosystems, Newark, DE).

Techniques: Expressing, Microarray, Staining, Software

Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 2 Specific knockdown of HER3 expression markedly inhibits TNBC cell proliferation and mammosphere formation in vitro and profoundly suppresses TNBC tumor growth in vivo. A and B, HCC1806, MDA-MB-468 (MDA-468), and MDA-MB-231 (MDA-231) cells were infected with lentivirus containing either control shRNA (shControl) or specific shRNA against HER3 (shHER3) for 24 h. The cells were then seeded in 96-well plates, incubated at a regular cell culture incubator, and monitored for growth via an IncuCyte system for 64 h. The cell growth curves were generated by GraphPad Prism9 software with the data obtained through IncuCyte scanning every 4 h (A). The cells were then subjected to mammosphere formation assays. After 14 days, rep resentative images of the mammospheres were shown and the number and size of those mammospheres were examined, quantified, and presented in the bar graphs (B). Data shows a representative of three independent experiments. Bars, SD. *, p < 0.05, **, p < 0.01, ***, p < 0.005. C, Luciferase-labelled HCC1806 cells were infected with lentivirus containing either control shRNA (shControl) or HER3 shRNA (shHER3) for 24 h. The HCC1806-shControl or HCC1806-shHER3 cells (5 × 105) were orthotopically inoculated into the left or right mammary fat pads of female nude mice, respectively (n = 4). In vivo bioluminescent imaging of the mammary tumors was obtained with the IVIS spectrum at day 1, 7, or 14 after cell inoculation. The graph showed the luciferase signal intensity of the mammary tumors obtained from the mice injected with HCC1806-shControl or HCC1806-shHER3 cells at day 1 and 14. A significant difference was observed using a two-sided Student’s t-test

Article Snippet: Specificity of 4A7 was further determined by ELISA with human HER3 His Tag (ER3-H5223) protein (ACROBiosystems, Newark, DE).

Techniques: Knockdown, Expressing, In Vitro, In Vivo, Infection, Control, shRNA, Incubation, Cell Culture, Generated, Software, Luciferase, Imaging, Injection

Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 3 Our newly developed anti-HER3 mAb 4A7 recognizes cell membrane HER3, and exhibits profound activity to block HRGβ1-, but not SDF-1-in duced TNBC cell migration. A, Our anti-HER3 mAb 4A7 was produced and purified as described in the materials and methods. 4A7 binding to mem brane HER3 on SKBR3.B3.1 cells was examined by flow cytometry analysis. Staining with 2nd Ab only was used as a negative control. 10,201-MM01, a commercially available anti-HER3 mAb was used a positive control. Quantification of the positive staining with either 10,201-MM01 or 4A7 from three independent experiments was shown in the bar graphs. B, Equal amount of total protein extracts of HCC1806 cells were either examined by western blot analyses of HER3 and IGF-1R (Input) or subjected to IP (IP: Ab) using a control IgG or 4A7 and followed by western blot analyses of HER3 and IGF-1R. C-D, The specificity of 4A7 against HER3 was confirmed by its capability to block HRGβ1-induced promotion of TNBC cell migration. HCC1806 cells seeded in 96-well plates were pre-incubated with mitomycin C (2 µg/ml) prior to the scratch assays. The cells were then untreated or treated with HRGβ1 (50ng/ml) or HRGβ1 plus 4A7 (20ug/ml) and monitored by an IncuCyte system for cell migration (C). The same migration assays were performed with MDA-MB-231 cells in the presence of HRGβ1 (50ng/ml) or SDF-1 (200ng/ml) or their combinations with 4A7 (D)

Article Snippet: Specificity of 4A7 was further determined by ELISA with human HER3 His Tag (ER3-H5223) protein (ACROBiosystems, Newark, DE).

Techniques: Membrane, Activity Assay, Blocking Assay, Migration, Produced, Purification, Binding Assay, Flow Cytometry, Staining, Negative Control, Positive Control, Western Blot, Control, Incubation

Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 4 Combinations of 4A7 and gefitinib potently inactivate HER3, EGFR, and their downstream signaling, and markedly induce growth inhibition and cell death in TNBC cells. A, The indicated TNBC cell lines cultured at normal condition were collected and subjected to western blot analyses of EGFR, p-EGFR (Y1068), HER3, and p-HER3 (Y1289). β-actin was used as a loading control. The relative signal intensity obtained via densitometry analyses was shown underneath of each image. B, Equal amount of total protein extracts of MDA-MB-468 (MDA-468) or HCC1806 cells were subjected to IP using a control IgG, anti-EGFR Ab (left), or anti-HER3 Ab (right), and followed by western blot analyses of HER3 and EGFR. C, MDA-MB-468 or HCC1806 cells at nor mal culture condition were treated with vehicle, 4A7, gefitinib, or the combinations of 4A7 and gefitinib for 24 h. The cells were collected and subjected to western blot assays of the indicated markers. Densitometry analyses of the signals were shown underneath, and the arbitrary numbers indicated the intensities of each image relative to the untreated controls, defined as 1.0. D, MDA-MB-468 or HCC1806 cells were plated in 96-well plates. After 24 h, the culture medium was replaced with fresh medium containing vehicle control (DMSO), gefitinib (4µmol/L) alone, or gefitinib (4µmol/L) plus 4A7 (20 µg/ml) (Gefi + 4A7), and incubated for additional 72 h. The percentages of surviving cells relative to controls, defined as 100% survival, were determined by cell growth/viability (MTS) assays. Data shows a representative of three independent experiments. Bars, SD. *, p < 0.05, **, p < 0.01. E, MDA-MB-468 or HCC1806 cells seeded in 6-well plates were treated with gefitinib (8µmol/L) or its combinations with 4A7 (20 µg/ml) for 48 h. The cells were subjected to the LIVE/ DEAD Cell Imaging assays. Green, live cells; red, dead cells. The ratio of dead/live cells was shown by in each sample. Data shows a representative of three independent experiments

Article Snippet: Specificity of 4A7 was further determined by ELISA with human HER3 His Tag (ER3-H5223) protein (ACROBiosystems, Newark, DE).

Techniques: Inhibition, Cell Culture, Western Blot, Control, Incubation, Imaging

Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 6 4A7 significantly enhances paclitaxel-mediated antitumor activity in TNBC tumor xenograft models. HCC1806-luc cells (5 × 105) were orthotopi cally inoculated into the mammary fat pads of nude mice to establish tumor xenografts. When tumor volume reached ~ 80mm3, the tumor-bearing mice received intraperitoneal injections of PBS, 4A7 (20 mg/kg), paclitaxel (6 mg/kg), or both 4A7 and paclitaxel (Pac + 4A7) (n = 5). After 6 treatments, mice were sacrificed. A, Tumor growth curves were plotted using average tumor volumes within each group at the indicated time points. A two-tailed student’s t-test was used for statistical analysis. Bars, SEM. *, P < 0.01. B, The mammary tumors were dissected and imaged as indicated (Left). The tumors were also measured for weight (Right). Bars, SD. *, p < 0.05, ***, p < 0.005. C and D, At end of the experiments, before mice were sacrificed, bioluminescent imaging of the mammary tumors was obtained with the IVIS spectrum (C). The luciferase signal intensity of the mammary tumors from the mice of each treatment group was plotted. Bars, SD. *, p < 0.05, **, p < 0.01. E, Formalin-fixed paraffin-embedded sections of tumors were analyzed with IHC staining for p-HER3 (Y1289), p-EGFR (Y1068), Ki67, or cleaved Caspase-3 (C-caspase-3). Scale bar, 70 μm. Quantification of IHC staining with ImageJ and ImageJ plugin IHC profiler was shown underneath. A two-way ANOVA tests were used for statistical analysis. Bars, SD. *, p < 0.05, ****, p < 0.001

Article Snippet: Specificity of 4A7 was further determined by ELISA with human HER3 His Tag (ER3-H5223) protein (ACROBiosystems, Newark, DE).

Techniques: Activity Assay, Two Tailed Test, Imaging, Luciferase, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 1 Elevated expression of HER3 is frequently observed in TNBC clinical samples and cell lines and associates with poor clinical outcomes in TNBC patients. A, IHC assays were performed to examine HER3 protein expression in a tissue microarray (TMA) of TNBC specimens (n = 125). Representative im ages of negative (-), low (+), medium (++), and high (+++) staining intensity of HER3 expression were shown. The case number in each group was shown underneath. B, The heatmap showed HER3 mRNA expression in CCLE breast cancer cell lines (http://software.broadinstitute.org/morpheus). Red colors indicated TNBC cell lines. C, Analyses of overall survival (OS) or relapse-free survival (RFS) of basal-like breast cancer patients were performed using Kaplan- Meier Plotter (https://kmplot.com/analysis/). The expression levels of HER3 mRNA were determined in TCGA-curated basal-like breast cancer datasets and used for log rank tests to compare the survival curves of those with high (red) or low (black) HER3 expression

Article Snippet: The TMA slides were deparaffinized and heated at 95oC for 30-40 min in Borg Decloaker, RTU (Biocare Medical, Pacheco, CA) for antigen retrieval, then blocked with a blocking sniper (Biocare Medical, Pacheco, CA), and then incubated with the anti-HER3 rabbit mAb (Cell Signaling Technology, cat# 12,708, 1: 400 dilution) at room temperature for 1 h. After washing with Tris Buffer Saline (pH 8.0), the slides were incubated with MACH 1 HRP Polymer detection kit (Biocare Medical) according to the manufacture’s instruction.

Techniques: Expressing, Microarray, Staining, Software

Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 2 Specific knockdown of HER3 expression markedly inhibits TNBC cell proliferation and mammosphere formation in vitro and profoundly suppresses TNBC tumor growth in vivo. A and B, HCC1806, MDA-MB-468 (MDA-468), and MDA-MB-231 (MDA-231) cells were infected with lentivirus containing either control shRNA (shControl) or specific shRNA against HER3 (shHER3) for 24 h. The cells were then seeded in 96-well plates, incubated at a regular cell culture incubator, and monitored for growth via an IncuCyte system for 64 h. The cell growth curves were generated by GraphPad Prism9 software with the data obtained through IncuCyte scanning every 4 h (A). The cells were then subjected to mammosphere formation assays. After 14 days, rep resentative images of the mammospheres were shown and the number and size of those mammospheres were examined, quantified, and presented in the bar graphs (B). Data shows a representative of three independent experiments. Bars, SD. *, p < 0.05, **, p < 0.01, ***, p < 0.005. C, Luciferase-labelled HCC1806 cells were infected with lentivirus containing either control shRNA (shControl) or HER3 shRNA (shHER3) for 24 h. The HCC1806-shControl or HCC1806-shHER3 cells (5 × 105) were orthotopically inoculated into the left or right mammary fat pads of female nude mice, respectively (n = 4). In vivo bioluminescent imaging of the mammary tumors was obtained with the IVIS spectrum at day 1, 7, or 14 after cell inoculation. The graph showed the luciferase signal intensity of the mammary tumors obtained from the mice injected with HCC1806-shControl or HCC1806-shHER3 cells at day 1 and 14. A significant difference was observed using a two-sided Student’s t-test

Article Snippet: The TMA slides were deparaffinized and heated at 95oC for 30-40 min in Borg Decloaker, RTU (Biocare Medical, Pacheco, CA) for antigen retrieval, then blocked with a blocking sniper (Biocare Medical, Pacheco, CA), and then incubated with the anti-HER3 rabbit mAb (Cell Signaling Technology, cat# 12,708, 1: 400 dilution) at room temperature for 1 h. After washing with Tris Buffer Saline (pH 8.0), the slides were incubated with MACH 1 HRP Polymer detection kit (Biocare Medical) according to the manufacture’s instruction.

Techniques: Knockdown, Expressing, In Vitro, In Vivo, Infection, Control, shRNA, Incubation, Cell Culture, Generated, Software, Luciferase, Imaging, Injection

Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 3 Our newly developed anti-HER3 mAb 4A7 recognizes cell membrane HER3, and exhibits profound activity to block HRGβ1-, but not SDF-1-in duced TNBC cell migration. A, Our anti-HER3 mAb 4A7 was produced and purified as described in the materials and methods. 4A7 binding to mem brane HER3 on SKBR3.B3.1 cells was examined by flow cytometry analysis. Staining with 2nd Ab only was used as a negative control. 10,201-MM01, a commercially available anti-HER3 mAb was used a positive control. Quantification of the positive staining with either 10,201-MM01 or 4A7 from three independent experiments was shown in the bar graphs. B, Equal amount of total protein extracts of HCC1806 cells were either examined by western blot analyses of HER3 and IGF-1R (Input) or subjected to IP (IP: Ab) using a control IgG or 4A7 and followed by western blot analyses of HER3 and IGF-1R. C-D, The specificity of 4A7 against HER3 was confirmed by its capability to block HRGβ1-induced promotion of TNBC cell migration. HCC1806 cells seeded in 96-well plates were pre-incubated with mitomycin C (2 µg/ml) prior to the scratch assays. The cells were then untreated or treated with HRGβ1 (50ng/ml) or HRGβ1 plus 4A7 (20ug/ml) and monitored by an IncuCyte system for cell migration (C). The same migration assays were performed with MDA-MB-231 cells in the presence of HRGβ1 (50ng/ml) or SDF-1 (200ng/ml) or their combinations with 4A7 (D)

Article Snippet: The TMA slides were deparaffinized and heated at 95oC for 30-40 min in Borg Decloaker, RTU (Biocare Medical, Pacheco, CA) for antigen retrieval, then blocked with a blocking sniper (Biocare Medical, Pacheco, CA), and then incubated with the anti-HER3 rabbit mAb (Cell Signaling Technology, cat# 12,708, 1: 400 dilution) at room temperature for 1 h. After washing with Tris Buffer Saline (pH 8.0), the slides were incubated with MACH 1 HRP Polymer detection kit (Biocare Medical) according to the manufacture’s instruction.

Techniques: Membrane, Activity Assay, Blocking Assay, Migration, Produced, Purification, Binding Assay, Flow Cytometry, Staining, Negative Control, Positive Control, Western Blot, Control, Incubation

Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 4 Combinations of 4A7 and gefitinib potently inactivate HER3, EGFR, and their downstream signaling, and markedly induce growth inhibition and cell death in TNBC cells. A, The indicated TNBC cell lines cultured at normal condition were collected and subjected to western blot analyses of EGFR, p-EGFR (Y1068), HER3, and p-HER3 (Y1289). β-actin was used as a loading control. The relative signal intensity obtained via densitometry analyses was shown underneath of each image. B, Equal amount of total protein extracts of MDA-MB-468 (MDA-468) or HCC1806 cells were subjected to IP using a control IgG, anti-EGFR Ab (left), or anti-HER3 Ab (right), and followed by western blot analyses of HER3 and EGFR. C, MDA-MB-468 or HCC1806 cells at nor mal culture condition were treated with vehicle, 4A7, gefitinib, or the combinations of 4A7 and gefitinib for 24 h. The cells were collected and subjected to western blot assays of the indicated markers. Densitometry analyses of the signals were shown underneath, and the arbitrary numbers indicated the intensities of each image relative to the untreated controls, defined as 1.0. D, MDA-MB-468 or HCC1806 cells were plated in 96-well plates. After 24 h, the culture medium was replaced with fresh medium containing vehicle control (DMSO), gefitinib (4µmol/L) alone, or gefitinib (4µmol/L) plus 4A7 (20 µg/ml) (Gefi + 4A7), and incubated for additional 72 h. The percentages of surviving cells relative to controls, defined as 100% survival, were determined by cell growth/viability (MTS) assays. Data shows a representative of three independent experiments. Bars, SD. *, p < 0.05, **, p < 0.01. E, MDA-MB-468 or HCC1806 cells seeded in 6-well plates were treated with gefitinib (8µmol/L) or its combinations with 4A7 (20 µg/ml) for 48 h. The cells were subjected to the LIVE/ DEAD Cell Imaging assays. Green, live cells; red, dead cells. The ratio of dead/live cells was shown by in each sample. Data shows a representative of three independent experiments

Article Snippet: The TMA slides were deparaffinized and heated at 95oC for 30-40 min in Borg Decloaker, RTU (Biocare Medical, Pacheco, CA) for antigen retrieval, then blocked with a blocking sniper (Biocare Medical, Pacheco, CA), and then incubated with the anti-HER3 rabbit mAb (Cell Signaling Technology, cat# 12,708, 1: 400 dilution) at room temperature for 1 h. After washing with Tris Buffer Saline (pH 8.0), the slides were incubated with MACH 1 HRP Polymer detection kit (Biocare Medical) according to the manufacture’s instruction.

Techniques: Inhibition, Cell Culture, Western Blot, Control, Incubation, Imaging

Journal: Cancer cell international

Article Title: HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

doi: 10.1186/s12935-023-03055-w

Figure Lengend Snippet: Fig. 6 4A7 significantly enhances paclitaxel-mediated antitumor activity in TNBC tumor xenograft models. HCC1806-luc cells (5 × 105) were orthotopi cally inoculated into the mammary fat pads of nude mice to establish tumor xenografts. When tumor volume reached ~ 80mm3, the tumor-bearing mice received intraperitoneal injections of PBS, 4A7 (20 mg/kg), paclitaxel (6 mg/kg), or both 4A7 and paclitaxel (Pac + 4A7) (n = 5). After 6 treatments, mice were sacrificed. A, Tumor growth curves were plotted using average tumor volumes within each group at the indicated time points. A two-tailed student’s t-test was used for statistical analysis. Bars, SEM. *, P < 0.01. B, The mammary tumors were dissected and imaged as indicated (Left). The tumors were also measured for weight (Right). Bars, SD. *, p < 0.05, ***, p < 0.005. C and D, At end of the experiments, before mice were sacrificed, bioluminescent imaging of the mammary tumors was obtained with the IVIS spectrum (C). The luciferase signal intensity of the mammary tumors from the mice of each treatment group was plotted. Bars, SD. *, p < 0.05, **, p < 0.01. E, Formalin-fixed paraffin-embedded sections of tumors were analyzed with IHC staining for p-HER3 (Y1289), p-EGFR (Y1068), Ki67, or cleaved Caspase-3 (C-caspase-3). Scale bar, 70 μm. Quantification of IHC staining with ImageJ and ImageJ plugin IHC profiler was shown underneath. A two-way ANOVA tests were used for statistical analysis. Bars, SD. *, p < 0.05, ****, p < 0.001

Article Snippet: The TMA slides were deparaffinized and heated at 95oC for 30-40 min in Borg Decloaker, RTU (Biocare Medical, Pacheco, CA) for antigen retrieval, then blocked with a blocking sniper (Biocare Medical, Pacheco, CA), and then incubated with the anti-HER3 rabbit mAb (Cell Signaling Technology, cat# 12,708, 1: 400 dilution) at room temperature for 1 h. After washing with Tris Buffer Saline (pH 8.0), the slides were incubated with MACH 1 HRP Polymer detection kit (Biocare Medical) according to the manufacture’s instruction.

Techniques: Activity Assay, Two Tailed Test, Imaging, Luciferase, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Nature communications

Article Title: ORMDL3 promotes eosinophil trafficking and activation via regulation of integrins and CD48

doi: 10.1038/ncomms3479

Figure Lengend Snippet: (a) ORMDL3 expression in BM-derived eosinophils cultured in medium alone (control) or medium containing IL-5, IL-3, RANTES-1 (100 ng/ml) or eotaxin-1 (Eot-1, 100 nM) for 2 h by RT-PCR (top) and qPCR (bottom). (b) ORMDL3 expression in eosinophils treated with IL-3 for 12 h by Western blot analysis followed by densitometry. Densitometry data is normalized against expression of β-actin used as the internal control. ORMDL3 expression in representative control and IL-3-treated eosinophil lysates is shown below bar graph. (c) Levels of ERK (1/2), phospho-ERK (1/2), p38 and phospho-p38 in eosinophils transfected with Control-GFP, ORMDL3 -GFP or left untreated by Western blot analysis. (d) NF-κB expression in DAPI-labeled Control-GFP- and ORMDL3 -GFP-transfected eosinophils by immunofluorescence staining with antibodies against phospho-NF-κB p65. Scale bar represents 10 µm. (e) Cytoskeletal arrangement in eosinophils transfected with Control-GFP or ORMDL3 -GFP by confocal microscopy after phalloidin staining. Scale bar represents 10 µm. (f) Rolling of murine eosinophils transfected with Control-GFP or ORMDL3 -GFP on rmVCAM-1-coated cover-slips under conditions of flow in vitro . Data shown in (a, top) and (c–e) is representative of two to three independent experiments with eosinophils from different mice. Combined data (mean ± SEM) from three experiments in triplicate in (a, bottom) and two experiments in duplicate in (f) is shown. In (b) , combined data (mean ± SD) from two out of three experiments with similar results is shown. *p < 0.01 in (a, bottom) compared to Control and < 0.02 in (f) compared to rolling on PBS; # p < 0.05 in (f) compared to Control-GFP. Statistical significance was determined by unpaired two-tailed Student’s t -test.

Article Snippet: Eosinophils cytocentrifuged onto glass slides were fixed, permeabilized, blocked and incubated over night at 4°C with polyclonal antibodies against phospho-NF-κB p65 (Santa Cruz Biotechnology, Inc., 1 μg/ml) followed by Rhodamine RedTM-X-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., 1:1000) , stained with DAPI and then examined by confocal microscopy.

Techniques: Expressing, Derivative Assay, Cell Culture, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Labeling, Immunofluorescence, Staining, Confocal Microscopy, In Vitro, Two Tailed Test