Journal: International Journal of Medical Sciences

Article Title: Effect of SDF-1 and CXCR4 gene variants on the development of diabetic kidney disease

doi: 10.7150/ijms.103186

Figure Lengend Snippet: Impact of rs1801157 and rs266085 genotypes on SDF-1 expression. Comparisons of SDF-1 (CXCL12) expression among (A) rs1801157 and (B) rs266085 genotypic groups in representative normal tissues based on data from the GTEx portal. p values were calculated among groups by one-way ANOVA.

Article Snippet: Biallelic discrimination for rs1801157 (assay ID: C_3223115_10, rs2297630 (assay ID: C_3223122_1), rs2839693 (assay ID: C_31777299_10), rs266085 (assay ID: C_1033724_30), rs2228014 (assay ID: C_27378716_10) and rs6430612 (assay ID: C_30721949_10) SNPs was carried out through the TaqMan assay (Applied Biosystems, Foster City, CA, USA), and genotypes were determined by SDS version 3.0 software.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: Knockdown of SChLAP1 inhibits the growth of triple negative breast cancer cells. (A) MDA-MB-231 and SKBR3 cells were transfected with control-siRNA or SChLAP1-siRNA. The knockdown of SChLAP1 was confirmed via reverse transcription-quantitative PCR analysis. A Cell Counting Kit-8 assay was performed to determine the viability of (B) MDA-MB-231 and (C) SKBR3 cells after transfection with siRNA-SChLAP1. (D) Colony formation ability was suppressed in MDA-MB-231 and SKBR3 cells upon knockdown of SChLAP1 (magnification, ×40). (E) Knockdown of SChLAP1 significantly promoted the apoptosis of both MDA-MB-231 and SKBR3 cells compared with cells expressing a control vector. ***P<0.001 vs. siRNA-Control. OD, optical density; siRNA, small interfering RNA; SChLAP1, second chromosome locus associated with prostate-1.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Knockdown, Transfection, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Expressing, Plasmid Preparation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: SChLAP1 acts as a sponge of miR-524-5p. (A) Predicted binding site of miR-524-5p on the sequence of SChLAP1. Luciferase activity of (B) MDA-MB-231 and (C) SKBR3 cells co-transfected with miR-524-5p mimics and WT-SChLAP1 was significantly decreased, while the activity of cells expressing miR-524-5p and Mut-SChLAP1 was not obviously affected. (D) Knockdown of SChLAP1 increased the expression of miR-524-5p in MDA-MB-231 and SKBR3 cells. (E) Expression of miR-524-5p in TNBC tissues was significantly downregulated compared with that of adjacent non-cancerous tissues. (F) Expression level of miR-524-5p was negatively correlated with SChLAP1 expression in TNBC tissues. ***P<0.001 vs. corresponding control. NC, negative control; miR, microRNA; WT, wild-type; Mut, mutant; TNBC, triple negative breast cancer; siRNA, small interfering RNA; SChLAP1, second chromosome locus associated with prostate-1.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Transfection, Expressing, Knockdown, Control, Negative Control, Mutagenesis, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: HMGA2 is a target of miR-524-5p in triple negative breast cancer. (A) MDA-MB-231 and SKBR3 cells were transfected with miR-524-5p mimic and mimic-NC, the expression of miR-524-5p was validated via reverse transcription-quantitative PCR. Cell Counting Kit-8 assay showed the decreased viability of (B) MDA-MB-231 and (C) SKBR3 cells induced by miR-524-5p overexpression. (D) Predicted binding sites of miR-524-5p in the 3′-UTR of HMGA2. Relative luciferase activities in (E) MB-231 and (F) SKBR3 cells co-transfected with miR-524-5p mimics or mimic-NC and luciferase reporter plasmid containing WT or Mut 3′-UTR of HMGA2. Overexpression of miR-524-5p decreased the (G) mRNA and (H) protein expression levels of HMGA2 in TNBC cells. The protein expression level of HMGA2 was semi-quantified by normalizing the level of GAPDH using ImageJ software. **P<0.01. ***P<0.001 vs. mimic-NC. UTR, untranslated region; NC, negative control; miR, microRNA; WT, wild-type; Mut, mutant; HMGA2, High Mobility Group AT-Hook 2; OD, optical density.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Over Expression, Binding Assay, Luciferase, Plasmid Preparation, Software, Negative Control, Mutagenesis

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: Reintroduction of HMGA2 reverses SChLAP1 knockdown-induced viability defects in TNBC cells. Knockdown of SChLAP1 decreased both the (A) mRNA and (B) protein expression levels of HMGA2 in MDA-MB-231 and SKBR3 cells. The protein expression level of HMGA2 was semi-quantified by normalizing the level of GAPDH using the ImageJ software. (C) Expression of Flag-tagged HMGA2 was detected via western blotting with anti-Flag antibody. Overexpression of HMGA2 significantly attenuated the effect of SChLAP1 knockdown on the viability of (D) MDA-MB-231 and (E) SKBR3 cells. (F) Co-transfection of HMGA2 promoted the colony formation of TNBC cells compared with cells expressing siRNA-SChLAP1 (magnification, ×40). ***P<0.001 vs. control group. HMGA2, High Mobility Group AT-Hook 2; TNBC, triple negative breast cancer; siRNA, small interfering RNA; SChLAP1, second chromosome locus associated with prostate-1; OD, optical density.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Knockdown, Expressing, Software, Western Blot, Over Expression, Cotransfection, Control, Small Interfering RNA

Journal: Cell Reports Medicine

Article Title: Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination

doi: 10.1016/j.xcrm.2021.100405

Figure Lengend Snippet: Immunogenicity of NVX-CoV2373 vaccine in rhesus macaques (A–C) Serum anti-spike (S) IgG titer (A), nasal wash (B), and bronchoalveolar lavage (BAL) (C) samples were collected 31/32 days after the first immunization and before challenge and analyzed for S-specific mucosal IgG (n = 4–5/group). (D) Pseudovirus-neutralizing titer (ID 50 ). (E) SARS-CoV-2 neutralizing-antibody titer (99% inhibition of cytopathic effect [99% CPE]) study day 31/32. (F) hACE2 receptor-blocking antibody titer (study day 31/32). The geometric mean titers (GMTs) are indicated by the white bars. Hollow arrows indicate prime/boosting with NVX-CoV2373. The error bars indicate the 95% confidence interval (95% CI). Individual animal values are indicated by colored symbols. A Student’s t test (unpaired, two-tailed) was used to compare antibody levels between groups immunized with one and two doses. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. The horizontal dashed lines indicate the LODs for each assay.

Article Snippet: After washing, 30 ng mL -1 histidine-tagged human ACE2 (Sino Biologics, Beijing, CHN) was added to wells for 1 h at room temperature.

Techniques: Inhibition, Blocking Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination

doi: 10.1016/j.xcrm.2021.100405

Figure Lengend Snippet:

Article Snippet: After washing, 30 ng mL -1 histidine-tagged human ACE2 (Sino Biologics, Beijing, CHN) was added to wells for 1 h at room temperature.

Techniques: Recombinant, Binding Assay, Blocking Assay, Mutagenesis, Software

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (A) Flow cytometry dot plot showing CD45 phosphatase activity versus CD27 expression on gated CD19 + human peripheral B cells. (B and C) CD45 phosphatase activity (B) and CD45 surface expression (C) of CD27 − (blue) and CD27 + B cells (red). Numbers in histograms represent CD45 activity (pCAP-SP1) (B) or CD45 surface expression (C) as the mean fluorescence intensity (MFI) ratio of CD27 + /CD27 − B cells. Bottom graphs: pCAP-SP1 or CD45 surface expression (MFI) in CD27 + relative to CD27 − B cells. (D) CD45 expression versus CD45 phosphatase activity in gated CD27 + MBCs; CD45 hi and CD45 lo expression gates are shown. (E) CD45 phosphatase activity and (F) CD45 expression in CD27 + MBCs expressing low (blue open histogram) or high (red open histogram) levels of surface CD45 compared to CD27 − B cells (filled blue histogram). Graphs show pCAP-SP1 or CD45 MFI relative to CD27 − B cells. n = 12. Related to . ****p < 0.0001.

Article Snippet: FITC Mouse monoclonal anti human CD27 (clone MT271) , Miltenyi Biotec , Cat# 130–093-184; RRID:AB_1036205.

Techniques: Flow Cytometry, Activity Assay, Expressing, Fluorescence

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: FITC Mouse monoclonal anti human CD27 (clone MT271) , Miltenyi Biotec , Cat# 130–093-184; RRID:AB_1036205.

Techniques: Negative Control, Recombinant, Purification, Staining, Gene Expression, Lysis, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cell Isolation, Software, Microscopy

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet: Endothelial, fibroblast and cardiomyocyte cell purification from mouse hearts (A) Scheme on the experimental procedures to isolate the indicated cell types from mouse hearts. (B) Heatmap of cell marker gene expression in RNA from isolated cardiomyocytes (CM), endothelial cells (EC) or fibroblasts (FB) after sham or the indicated time point after TAC surgery. (C) Cardiac endothelial cells and fibroblasts were isolated from mouse hearts and stained for the endothelial markers CD31 and CD102, for the leukocyte marker CD45, and the fibroblast marker Mefsk4. Subsequently, flow cytometric analyses were performed and representative results are shown here. The numbers indicated in each quadrant indicates the percentage of cells localized in that particular quadrant. (D) RNA from the different cell types after sham or 1 and 8 weeks after TAC was subjected to RNA sequencing. The differences in overall gene expression patterns were visualized by a principal component analysis.

Article Snippet: CD45 Antibody, anti-mouse , Miltenyi Biotec , Clone 30F11.

Techniques: Purification, Marker, Gene Expression, Isolation, Staining, RNA Sequencing

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet:

Article Snippet: CD45 Antibody, anti-mouse , Miltenyi Biotec , Clone 30F11.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Magnetic Beads, Recombinant, Clinical Proteomics, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet: Endothelial, fibroblast and cardiomyocyte cell purification from mouse hearts (A) Scheme on the experimental procedures to isolate the indicated cell types from mouse hearts. (B) Heatmap of cell marker gene expression in RNA from isolated cardiomyocytes (CM), endothelial cells (EC) or fibroblasts (FB) after sham or the indicated time point after TAC surgery. (C) Cardiac endothelial cells and fibroblasts were isolated from mouse hearts and stained for the endothelial markers CD31 and CD102, for the leukocyte marker CD45, and the fibroblast marker Mefsk4. Subsequently, flow cytometric analyses were performed and representative results are shown here. The numbers indicated in each quadrant indicates the percentage of cells localized in that particular quadrant. (D) RNA from the different cell types after sham or 1 and 8 weeks after TAC was subjected to RNA sequencing. The differences in overall gene expression patterns were visualized by a principal component analysis.

Article Snippet: CD31 Antibody, anti-mouse , Miltenyi Biotec , Clone 390.

Techniques: Purification, Marker, Gene Expression, Isolation, Staining, RNA Sequencing

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet:

Article Snippet: CD31 Antibody, anti-mouse , Miltenyi Biotec , Clone 390.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Magnetic Beads, Recombinant, Clinical Proteomics, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: APC-Vio770 Recombinant human anti B220(clone REA755) , Miltenyi Biotec , Cat# 130–110-849; RRID:AB_2658286.

Techniques: Negative Control, Recombinant, Purification, Staining, Gene Expression, Lysis, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cell Isolation, Software, Microscopy

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Coxsackievirus B3 Infection Early in Pregnancy Induces Congenital Heart Defects Through Suppression of Fetal Cardiomyocyte Proliferation

doi: 10.1161/JAHA.120.017995

Figure Lengend Snippet: A , Top 5 differentially regulated Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways in heart tissue from embryonic day (E) 14 fetuses infected with CVB3 at E9. B , Comparison of RNA‐sequencing (black dot, n=4) and quantitative reverse transcription–polymerase chain reaction (white dot, n=4) data for upregulated genes within the pathways (transforming growth factor [TGF]‐β1, BMP2 [bone morphogenetic protein 2], Smad1 , Smad5 , and Smad9 ), normalized to Gapdh expression in heart tissue from E14 fetuses infected with CVB3 at E9. C , pHH3‐stained cells (individual data points as white dots and mean as black dot±SD), indicating lower cell proliferation in the CVB3‐infected fetal hearts (E11 and E17) compared with uninfected controls (n=4 for each gestational age). D and E , Immunofluorescence images showing 4′,6‐diamidino‐2‐phenylindole (DAPI)–stained DNA (blue), MF20‐stained cardiomyocytes (green), and pHH3‐stained proliferating cells (red) in the E11 control and infected fetuses ( D ) and E17 control and infected fetuses ( E ) (magnified images of the white box in the panels on the left). The number of stained cells was determined using ImageJ software. The images are representative of 4 biological replicates. Bar=100 µm. ECM indicates extracellular matrix.

Article Snippet: Samples were blocked for 1 hour at room temperature with 10% fetal bovine serum and then incubated overnight at 4°C with primary antibodies PHH3 (Millipore 06‐570) to identify mitotic cells, MF20 (Developmental Studies Hybridoma Bank, MF20) to identify myocytes, and Mouse Anti‐Enterovirus Clone 5‐D8/1 (Dako M7064) to identify CVB VP1 (viral capsid protein) region.

Techniques: Infection, Comparison, RNA Sequencing, Reverse Transcription, Polymerase Chain Reaction, Expressing, Staining, Immunofluorescence, Control, Software

Journal: Cell Reports Medicine

Article Title: Vaccine-induced ICOS + CD38 + circulating Tfh are sensitive biosensors of age-related changes in inflammatory pathways

doi: 10.1016/j.xcrm.2021.100262

Figure Lengend Snippet:

Article Snippet: Anti-human Survivin (REA459) , Miltenyi , Cat # 130-106-741; RRID: AB_2653606.

Techniques: Recombinant, Staining, Gene Expression, Software

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: CyTOF antibody panel

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques:

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Selected genes involved in Tfh cell biology

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques: Activation Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Distinct CD4 + T cell subsets contribute to the generation of Tfh with heterogeneous functional profiles (A) Mean fluorescence intensity of the CXCR5 marker expressed by CXCR5 + PD-1 + cells derived from (1) naive, (2) MemPD-1 neg , (3) MemPD-1 neg and Tfh. (B) Frequency of IL-21- and/or IFNγ-positive cells among CXCR5 + PD-1 + cells at day 3. (C – E) Representative flow plots showing CXCR3, ICOS, and CD40L expression by CXCR5 + PD-1 + cells (left panel) and frequency of CXCR3-, ICOS-, and CD40L-positive cells among CXCR5 + PD-1 + cells at day 3 (right panel). (F) Ex vivo cells or their respective Tfh D3 counterparts obtained after 3 days of splenocyte culture were co-cultured with autologous B cells for 7 days. (G) Box plots represent the frequency of CD27 + CD38 + cells among CD19 + cells, the concentration of total immunoglobulins and the absolute number of live B cells after co-culture. (H) Quantification of IgG1, IgG4, and IgA in the co-culture supernatants. Each symbol (A–H) represents an individual donor. (A–H) A Wilcoxon matched pairs test was performed, ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.001.

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques: Functional Assay, Fluorescence, Marker, Derivative Assay, Expressing, Ex Vivo, Cell Culture, Concentration Assay, Co-Culture Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet:

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques: Cell Analysis, Purification, Virus, Recombinant, Blocking Assay, Antibody Labeling, Transfection, Software

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Mouse anti-mouse CD45.1 (FITC) , Miltenyi Biotec , Cat# 130–124-211; RRID: AB_2857674.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Molecular Biology of the Cell

Article Title: Cross-talk between androgen receptor/filamin A and TrkA regulates neurite outgrowth in PC12 cells

doi: 10.1091/mbc.E14-09-1352

Figure Lengend Snippet: Role of FlnA and its association with AR in neurite outgrowth induced by R1881 or NGF. PC12 cells were used. (A) Growing cells on polylysine-coated coverslips were transfected with FlnA siRNA or nt siRNA. Purified pEGFP plasmid (Amaxa) was included to help identification of transfected cells. Cells were made quiescent and then left unchallenged or challenged for 48 h with R1881 (10 nM) or NGF (100 ng/ml). In GFP-expressing cells, neurite outgrowth was analyzed by contrast phase microscopy and expressed as percentage of transfected cells. Several coverslips were analyzed, and data from at least 150 scored cells for each coverslip were collected and are graphically shown. Inset in A shows FlnA protein levels detected by Western blot of lysate proteins from PC12 cells transfected with nt or FlnA siRNA. After silencing, residual FlnA expression was ∼20%, as calculated by ImageJ software. (B) Quiescent cells were left untreated or treated for 5 min with the indicated compounds: R1881 at 10 nM, bicalutamide (Bic) at 10 μM, and S peptide at 1 nM. Lysate proteins (2 mg/ml protein) were immunoprecipitated with nonspecific (ctrl Ab) or anti-AR antibodies (C-19 Ab). Proteins in immune complexes were analyzed using antibodies against the indicated proteins. The C-19 Ab was used to detect AR. (C) Quiescent cells on polylysine-coated coverslips were left untreated or treated for 48 h with 10 nM R1881 or 100 ng/ml NGF in the absence or presence of S peptide (1 nM). Neurite outgrowth in plastic-plated PC12 cells was evaluated and is expressed as percentage of total cells. In A and C, means and SEM are shown. In C, the statistical significance of results was also evaluated by paired t test. The difference in neurite outgrowth was significant (* p < 0.05) only between cells challenged with R1881 or NGF and unstimulated cells. (D) Quiescent cells were left untreated or treated for the indicated times with 10 nM R1881 (top) or 100 ng/ml NGF (bottom). Lysate proteins were analyzed using antibodies against the indicated proteins.

Article Snippet: In PC12 cells, FlnA was stained using diluted (1:30 in PBS) goat polyclonal anti-FlnA (Ab11074; Abcam, Cambridge, UK).

Techniques: Transfection, Purification, Plasmid Preparation, Expressing, Microscopy, Western Blot, Software, Immunoprecipitation

Journal: Molecular Biology of the Cell

Article Title: Cross-talk between androgen receptor/filamin A and TrkA regulates neurite outgrowth in PC12 cells

doi: 10.1091/mbc.E14-09-1352

Figure Lengend Snippet: R1881 or NGF induces extranuclear AR/FlnA complex in PC12 cells. Quiescent PC12 cells were left untreated (ctrl) or treated for 5 min with 10 nM R1881 (R1881) or 100 ng/ml NGF (NGF). (A) Cells on coverslips were visualized by IF for AR and Fln A. Images captured by confocal microscope show the staining of AR (green) and Fln A (red). Right, merged images. Confocal microscopy analysis is representative of three independent experiments. Bar, 10 μm. (B) Quantification and statistical analysis of these experiments. AR/FlnA colocalization ratio was calculated as described in Materials and Methods . Data from several independent experiments were analyzed. Means and SEM are shown; n represents the number of experiments. The statistical significance of results was also evaluated by paired t test. The difference in AR/FlnA colocalization ratio between unstimulated (ctrl) and R1881-stimulated cells was significant ( p < 0.001). Also significant ( p < 0.001) was the difference in AR/FlnA colocalization ratio between control (ctrl) and NGF-stimulated cells.

Article Snippet: In PC12 cells, FlnA was stained using diluted (1:30 in PBS) goat polyclonal anti-FlnA (Ab11074; Abcam, Cambridge, UK).

Techniques: Microscopy, Staining, Confocal Microscopy, Control

Journal: Molecular Biology of the Cell

Article Title: Cross-talk between androgen receptor/filamin A and TrkA regulates neurite outgrowth in PC12 cells

doi: 10.1091/mbc.E14-09-1352

Figure Lengend Snippet: Model based on our experimental findings. Androgen or NGF induces association of TrkA/β1 integrin/AR/FlnA /PI3-K δ complex, which in turn triggers Rac activation and neuritogenesis in cells challenged with androgens or NGF. Images show the cytoskeleton changes induced by 10 nM R1881 in quiescent PC12 cells.

Article Snippet: In PC12 cells, FlnA was stained using diluted (1:30 in PBS) goat polyclonal anti-FlnA (Ab11074; Abcam, Cambridge, UK).

Techniques: Activation Assay