Journal: The EMBO Journal

Article Title: Micro RNA ‐34/449 controls mitotic spindle orientation during mammalian cortex development

doi: 10.15252/embj.201694056

Figure Lengend Snippet: A Quantification of mRNA expression by RT–qPCR of candidate miR‐34/449 targets that might regulate spindle orientation in E14 cortex samples from miR‐34/449 KO mice compared with littermate controls (Het). Expression was normalized (norm.) to phosphoglycerate kinase (PGK) mRNA levels. Significance was tested by Welch's t ‐test: ** P = 0.009756 (JAM‐A (Het) vs. JAM‐A (KO)), all the rest Het vs. KO comparisons P = n.s. ( n = 4 cortices per genotype group, 2 independent litters). B RT–qPCR quantification of JAM‐A mRNA expression in HeLa cells 48 h after transfection of miR‐449a mimic compared to cells transfected with nontargeting negative control siRNA. JAM‐A mRNA expression was normalized (norm.) to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA levels. *** P = 0.0001218 (Neg. control vs. miR‐449a mimic) ( n = 9 measurements from 3 independent replicates). C, D Mouse J110 cells were transfected with either miR‐449a mimic or a negative control mimic. Whole‐cell extracts were subjected to immunoblot analysis to detect endogenous JAM‐A and actin. Actin was used to normalize JAM‐A expression (norm.) as loading control. * P = 0.04454 (Neg. control vs. miR‐449a mimic) ( n = 4 independent wells, 2 independent transfections). E, F HeLa cells were cotransfected with either miR‐449a mimic or a negative control mimic, together with JAM‐A–GFP fusion protein and GFP. Whole‐cell extracts were subjected to immunoblot analysis to detect GFP. GFP was used to normalize (norm.) by transfection efficiency as loading control. ** P = 0.001176 (JAM‐A WT Neg. control vs. miR‐449a mimic) ( n = 9 independent wells, 4 independent transfections). G, H Confocal images of coronal sections from E14 brains of wild‐type mice stained with anti‐JAM‐A antibody (green), phalloidin to label actin (gray), and DAPI to label cell nuclei (blue). Scale bars: 10 μm (G), 5 μm (H). I Quantification of JAM‐A protein expression in the ventricular zone of E14 brain coronal sections from miR‐34/449 KO mice compared with littermate controls (Het) by immunofluorescence. Actin (phalloidin signal) was used to normalize (norm.) the expression levels of JAM‐A. * P = 0.04842 ( n = 5 (KO) or 7 (het) cortices per genotype group, 3 independent litters). Data information: Red bars indicate the median. Statistical significance was tested by Welch's t ‐test. Source data are available online for this figure.

Article Snippet: Western blotting was performed by standard methods using antibodies against GFP (1:500, AB clone 2B6, MFPL Monoclonal Antibody Facility, Vienna), Cas9 (1:500, MFPL Monoclonal Antibody Facility, Vienna), JAM‐A (1:100, AB H202‐106 batch g3013, Santa Cruz Biotechnology), and actin (1:10,000, AB clone C4, Millipore).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Negative Control, Western Blot, Staining, Immunofluorescence

Journal: The EMBO Journal

Article Title: Micro RNA ‐34/449 controls mitotic spindle orientation during mammalian cortex development

doi: 10.15252/embj.201694056

Figure Lengend Snippet: A, B A schematic representation of the predicted miR‐34/449 binding sites in the mouse and human JAM‐A CDS, respectively. C Schematic representation of the GFP–JAM‐A fusion reporters containing WT (WT) or mutant (MUT) JAM‐A CDS for the binding of miR‐34/449 miRNA family.

Article Snippet: Western blotting was performed by standard methods using antibodies against GFP (1:500, AB clone 2B6, MFPL Monoclonal Antibody Facility, Vienna), Cas9 (1:500, MFPL Monoclonal Antibody Facility, Vienna), JAM‐A (1:100, AB H202‐106 batch g3013, Santa Cruz Biotechnology), and actin (1:10,000, AB clone C4, Millipore).

Techniques: Binding Assay, Mutagenesis

Journal: The EMBO Journal

Article Title: Micro RNA ‐34/449 controls mitotic spindle orientation during mammalian cortex development

doi: 10.15252/embj.201694056

Figure Lengend Snippet: Confocal time‐lapse images of metaphase HeLa cells stably expressing a centriole marker (centrin‐2–eGFP; green) and a microtubule marker (α‐tubulin–mRFP; red), 48 h after transfection of miR‐449a mimic, JAM‐A siRNA, or nontargeting negative control siRNA. Scale bar, 10 μm. Quantification of spindle rotation for cells as shown in (A). Each dot represents a single cell. Red bar indicates median. Significance was tested by pairwise t ‐test with Bonferroni correction. *** P ‐value = 2e‐11 (Neg. control vs. miR449 mimic), * P ‐value = 0.01928 (Neg. control vs. JAM‐A siRNA). Cumulative histograms of mitotic duration from nuclear envelope breakdown until anaphase onset, measured for cells as shown in (A); n ≥ 20 cells for all conditions, 2 independent replicates. Confocal time‐lapse images of metaphase HeLa cells stably expressing a centriole marker (centrin‐2–eGFP; green), a microtubule marker (α‐tubulin–mRFP; red), and mouse JAM‐A, 48 h after transfection of miR‐449a mimic, JAM‐A siRNA, or nontargeting negative control siRNA. Scale bar, 10 μm. Quantification of spindle rotation for cells as shown in (D). Each dot represents a single cell. Red bar indicates median. Significance was tested by pairwise t ‐test with Bonferroni correction. n.s., P ‐value = 0.17492 (Neg. control vs. miR449 mimic). Cumulative histograms of mitotic duration from nuclear envelope breakdown until anaphase onset, measured for cells as shown in (D); n ≥ 20 cells for all conditions, 2 independent replicates. Source data are available online for this figure.

Article Snippet: Western blotting was performed by standard methods using antibodies against GFP (1:500, AB clone 2B6, MFPL Monoclonal Antibody Facility, Vienna), Cas9 (1:500, MFPL Monoclonal Antibody Facility, Vienna), JAM‐A (1:100, AB H202‐106 batch g3013, Santa Cruz Biotechnology), and actin (1:10,000, AB clone C4, Millipore).

Techniques: Stable Transfection, Expressing, Marker, Transfection, Negative Control

Journal: The EMBO Journal

Article Title: Micro RNA ‐34/449 controls mitotic spindle orientation during mammalian cortex development

doi: 10.15252/embj.201694056

Figure Lengend Snippet: Confocal time‐lapse images of metaphase HeLa cells stably expressing a centriole marker (centrin‐2–eGFP; green), a microtubule marker (α‐tubulin–mRFP; red), and Cas9, 48 h after transfection of miR‐449a mimic, JAM‐A siRNA, or nontargeting negative control siRNA. Scale bar, 10 μm. Quantification of spindle rotation for cells as shown in (A). Each dot represents a single cell. Red bar indicates median. Significance was tested by pairwise t ‐test with Bonferroni correction. *** P ‐value = 7.9e‐12 (Neg. control vs. miR449 mimic, under JAM‐A overexpression). Cumulative histogram of mitotic duration from nuclear envelope breakdown until anaphase onset, measured for cells as shown in (A); n ≥ 20 cells for all conditions, 2 independent replicates. Whole‐cell extracts from the HeLa Kyoto stable cell lines used in Figs and expressing JAM‐A and Cas9 were subjected to immunoblot analysis to detect mouse JAM‐A, Cas9, and actin as an endogenous control protein. Source data are available online for this figure.

Article Snippet: Western blotting was performed by standard methods using antibodies against GFP (1:500, AB clone 2B6, MFPL Monoclonal Antibody Facility, Vienna), Cas9 (1:500, MFPL Monoclonal Antibody Facility, Vienna), JAM‐A (1:100, AB H202‐106 batch g3013, Santa Cruz Biotechnology), and actin (1:10,000, AB clone C4, Millipore).

Techniques: Stable Transfection, Expressing, Marker, Transfection, Negative Control, Over Expression, Western Blot

Journal: Oncotarget

Article Title: Junctional adhesion molecule-A is overexpressed in advanced multiple myeloma and determines response to oncolytic reovirus.

doi: 10.18632/oncotarget.5753

Figure Lengend Snippet: Figure 3: JAM-A regulates reovirus sensitivity in MM. A. shRNA-mediated knockdown of JAM-A in RPMI-8226 cells. Cells were infected with JAM-A lentiviral shRNA and knockdown was determined by immunoblotting. B. Cells with silenced JAM-A are significantly less sensitive to Reolysin. Cells infected with control or JAM-A shRNA were treated with Reolysin for 72 h. Cell viability was measured by MTT assay. Mean ± SD, n = 3, *p < 0.05 compared to shRNA control treated cells. C. Cells with decreased JAM-A levels are resistant to Reolysin-mediated apoptosis. RPMI-8226 cells were treated with 30 PFU/Cell Reolysin for 48 h. Active caspase-3 levels were measured by fluorescent staining and flow cytometry. Mean ± SD, n = 3, *p < 0.05 compared to shRNA control treated cells. D. Overexpression of JAM-A in OPM-2 cells. JAM-A was transfected into OPM-2 cells and immunoblotting confirmed increased expression at 72 h following transfection. E. Forced expression of JAM-A sensitizes OPM-2 cells to Reolysin. Vector control and JAM-A transfected cells were treated with the indicated concentrations of Reolysin for 72 h. Cell viability was measured by MTT assay. Mean ± SD, n = 3, *p < 0.05 compared to Vector control treated cells. F. JAM-A overexpression sensitizes OPM-2 cells to Reolysin-mediated apoptosis. OPM-2 cells were treated with 30 PFU/Cell Reolysin for 48 h and apoptosis was measured by active caspase-3 staining and flow cytometry. Mean ± SD, n = 3, *p < 0.05 compared to Vector control treated cells.

Article Snippet: Gene expression profiling data under accession number GSE2658 were collected from a publicly available website that includes 351 newly diagnosed patients with MM who participated in the Total Therapy 2 (TT2) clinical trial, 44 patients with monoclonal gammopathy of unknown significance (MGUS), and 22 normal plasma cell samples that were obtained from healthy donors [45, 46]. shRNA knockdown of JAM-A MM cells were infected with a lentivirus encoding a short hairpin RNA (shRNA) sequence specific for JAM-A or an empty vector (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: shRNA, Knockdown, Infection, Western Blot, Control, MTT Assay, Staining, Flow Cytometry, Over Expression, Transfection, Expressing, Plasmid Preparation