Journal: Science Advances

Article Title: Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration

doi: 10.1126/sciadv.add1101

Figure Lengend Snippet: ( A ) Representative dot plots show the immune profile in the brains of young and middle-aged Apoe , h APOE3 , and h APOE4 , quantified on the right. Representative histograms depict the relative level of cell granularity ( B ), autofluorescence ( C ), lipid accumulation ( D ), CD68 ( E ), and Lamp1 ( F ) protein expression and intracellular cytokine production of TNF ( G ) in CD45 int CD11b + microglia across ages and genotypes. ( H ) Ex vivo neuronal engulfment assay shows a significant increase in the percent of microglia that phagocytized live SLICK-YFP neurons, quantified on the right. Validation of internalized myelinated cortical neurons was performed using intracellular detection of anti-YFP ( I ) and anti-myelin CNPase ( J ) in phagocytic (SLICK-YFP + ) and nonphagocytic (SLICK-YFP − ) microglial populations within the same brain. N = 6 to 7 per group. MA, middle-aged; Y, young; ns, not significant.. Data were analyzed using one-way analysis of variance (ANOVA) with Bonferroni post hoc correction for multiple comparisons (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: Cells were washed twice in 500 μl of permeabilization/wash buffer (BD Biosciences) and resuspended in an intracellular antibody cocktail containing cytokine antibodies [MMP-9-R-PE (StressMarq Biosciences, SMC-396D), TNF-PE-Cy7 (eBioscience, MP6-XT22), IL-1α–PE (BioLegend, ALF-161), and IL-1β–PerCP–eF710 (eBioscience, NJTEN3) and fixed.

Techniques: Expressing, Ex Vivo

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer

doi: 10.3390/ijms241914847

Figure Lengend Snippet: MMP9 and miR-21 gene editing with CRISPR-Cas9. ( A ) DNA sequencing of the PX-330 plasmid with the sequence inserts for MMP9 (sgRNA1 and sgRNA2) at the beginning of MMP9 Exon 1 on chromosome 20q ( left ) and DNA sequencing of the sequence inserts for miR-21 (sgRNA 1 and sgRNA2), located in two regions of chromosome 17 ( right ). ( B ) miR-21 gene expression in samples edited with miR-21 sgRNA 1 and their respective control transfected with the plasmid without any insert (Scramble) in the PC-3 cell line. ( C , D ) MMP9 and RECK gene expression in samples edited with CRISPR-Cas9 MMP9, sgRNAs 1 and 2 or miR-21 sgRNA1 compared with the scramble control in the PC-3 cell line. ( E ) miR-21 gene expression in samples edited with CRISPR-Cas9 miR-21 sgRNA 1 compared to the scramble control in the DU145 cell line. ( F , G ) MMP9 and RECK gene expression in samples edited with CRISPR-Cas9 MMP9 sgRNAs 1 and 2 or miR-21 sgRNA1 compared to Scramble in the DU145 cell line. Statistical significance set at p < 0.05.

Article Snippet: Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed 3 times with PBS and subjected to blocking solution with 3% BSA in PBS for 1 h. The MMP9 (1:150, Boster M00139) and RECK (1:100, Santa Cruz SC373929) antibodies were incubated in PBS containing 1% BSA overnight at 4 °C.

Techniques: CRISPR, DNA Sequencing, Plasmid Preparation, Sequencing, Gene Expression, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer

doi: 10.3390/ijms241914847

Figure Lengend Snippet: MMP9 and RECK protein expression in MMP9 and miR-21 CRISPR-Cas9-edited metastatic PCa cell lines. ( A ) Western blot analysis of MMP9 protein content in PC-3 and DU145 cells edited with MMP9 sgRNA 1/2 or miR-21 sgRNA1. ( B , C ) Protein immunofluorescence of colocalized MMP9 (green) and RECK (red) in samples edited with MMP9 sgRNA 1/2 or miR-21 sgRNA1 in both PC-3 and DU145 cells.

Article Snippet: Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed 3 times with PBS and subjected to blocking solution with 3% BSA in PBS for 1 h. The MMP9 (1:150, Boster M00139) and RECK (1:100, Santa Cruz SC373929) antibodies were incubated in PBS containing 1% BSA overnight at 4 °C.

Techniques: Expressing, CRISPR, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer

doi: 10.3390/ijms241914847

Figure Lengend Snippet: Gene expression of miR-21 targets in metastatic PCa cell lines. Gene expression of ( A ) MARKS, ( B ) BTG2, and ( C ) PDCD4 in MMP9 and miR-21 CRISPR-Cas9-edited PC-3 cells, and ( D ) MARKS, ( E ) BTG2, and ( F ) PDCD4 in MMP9 and miR-21 CRISPR-Cas9-edited DU145 cells.

Article Snippet: Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed 3 times with PBS and subjected to blocking solution with 3% BSA in PBS for 1 h. The MMP9 (1:150, Boster M00139) and RECK (1:100, Santa Cruz SC373929) antibodies were incubated in PBS containing 1% BSA overnight at 4 °C.

Techniques: Gene Expression, CRISPR

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer

doi: 10.3390/ijms241914847

Figure Lengend Snippet: Gene expression of CDH1, integrins, BAX, and mTOR in metastatic PCa lines. Gene expression of ( A ) CDH1 cadherin, integrins ( B ) ITGB3 and ( C ) ITGB1, ( D ) BAX and ( E ) mTOR in MMP9 sgRNA 1/2- and miR-21 sgRNA1 CRISPR-Cas9-edited PC-3 cells. Gene expression of ( F ) CDH1 cadherin, integrins ( G ) ITGB3 and ( H ) ITGB1, ( I ) BAX and ( J ) mTOR in MMP9 sgRNA 1/2- and miR-21 sgRNA1 CRISPR-Cas9-edited DU145 cell line.

Article Snippet: Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed 3 times with PBS and subjected to blocking solution with 3% BSA in PBS for 1 h. The MMP9 (1:150, Boster M00139) and RECK (1:100, Santa Cruz SC373929) antibodies were incubated in PBS containing 1% BSA overnight at 4 °C.

Techniques: Gene Expression, CRISPR

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer

doi: 10.3390/ijms241914847

Figure Lengend Snippet: Flow cytometry for assessing proliferation and apoptosis in the metastatic PC-3 and DU145 cell lines. ( A ) Labeling MMP9 sgRNA1/2- and miR-21 sgRNA1 CRISPR-Cas9-edited PC-3 cells with a ki67 antibody to evaluate cell proliferation rate. ( B – D ) Labeling MMP9 sgRNA1/2 and miR-21 sgRNA1 CRISPR-Cas9-edited PC-3 cells with annexin-5 and 7-AAD to calculate the percentage of cells in the early and late stages of apoptosis and total apoptosis. ( E ) Labeling MMP9 sgRNA1/2 and miR-21 sgRNA1 CRISPR-Cas9-edited DU145 cells with a ki67 antibody to evaluate cell proliferation rate. ( F – H ) Labeling MMP9 sgRNA1/2 and miR-21 sgRNA1 CRISPR-Cas9-edited DU145 cells with annexin-5 and 7-AAD to calculate the percentage of cells in the early and late stages of apoptosis and total apoptosis.

Article Snippet: Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed 3 times with PBS and subjected to blocking solution with 3% BSA in PBS for 1 h. The MMP9 (1:150, Boster M00139) and RECK (1:100, Santa Cruz SC373929) antibodies were incubated in PBS containing 1% BSA overnight at 4 °C.

Techniques: Flow Cytometry, Labeling, CRISPR

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer

doi: 10.3390/ijms241914847

Figure Lengend Snippet: Transwell chamber invasion assay with metastatic ( A ) PC-3 and ( B ) DU145 CRISPR-Cas9 MMP9 sgRNA1/2- and miR-21 sgRNA1-edited cell lines.

Article Snippet: Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed 3 times with PBS and subjected to blocking solution with 3% BSA in PBS for 1 h. The MMP9 (1:150, Boster M00139) and RECK (1:100, Santa Cruz SC373929) antibodies were incubated in PBS containing 1% BSA overnight at 4 °C.

Techniques: Invasion Assay, CRISPR