Journal: bioRxiv

Article Title: Inducible overexpression of zebrafish microRNA-722 suppresses chemotaxis of human neutrophil like cells

doi: 10.1101/578583

Figure Lengend Snippet: (A) Schematics of the experimental flow for the inducible expression of a microRNA and a reporter. The stable HL-60 cell lines over expressing miR-722 or the vector control were differentiated with 1.3 % DMSO for 6 days and treated with 1 µg/ml doxycycline from day 4 or left untreated. (B) Levels of pre-miR-722 , miR-722 , MIR-223 , and LET7 in vector or miR-722 expressing dHL-60 lines ± doxycycline (DOX). Messenger RNAs were normalized to GAPDH and miRNAs were normalized to U6 . Results are presented as mean ± s.d. from three independent experiments. **, p < 0.01, Mann–Whitney test. (C, D) Histogram of CD11b expression (C) and Annexin V staining (D) of vector and miR-722 expressing dHL-60 with (blue) or without (green) DOX. Positive cells are gated based on the isotype control and percentages of positive cells are labeled in corresponding color. Images are representative of three independent experiments.

Article Snippet: Plasmids are available on Addgene: pCDNA miR-722-Dendra (#97163), pcDNA3.1 Dendra2 (#103967), pSi-check2-hRac2 3′UTR (#97160), pSi-check2-hRac2 -mut 3′UTR (#97161), plix4.03-miR-722 (#97140), plix4.03-vector (#97141).

Techniques: Expressing, Plasmid Preparation, MANN-WHITNEY, Staining, Labeling

Journal: bioRxiv

Article Title: Inducible overexpression of zebrafish microRNA-722 suppresses chemotaxis of human neutrophil like cells

doi: 10.1101/578583

Figure Lengend Snippet: (A) Volcano blot of DEGs with significant changes in expression upon doxycycline induced miR-722 expression. Red: down-regulated differentially expressed genes (DEGs); cyan: up-regulated DEGs. (B) Significant enrichment of human homologues of miR-722 predicted target genes in the down-regulated DEGs. P < 0.003, hypergeometric distribution. (C, D, E, F) Clusters of the genes in the pathways significantly altered upon miR-722 overexpression. Number of the genes with significant expression changes in each pathway are labeled in each column. q-values, FDR-adjusted p-values as described in the Methods section.

Article Snippet: Plasmids are available on Addgene: pCDNA miR-722-Dendra (#97163), pcDNA3.1 Dendra2 (#103967), pSi-check2-hRac2 3′UTR (#97160), pSi-check2-hRac2 -mut 3′UTR (#97161), plix4.03-miR-722 (#97140), plix4.03-vector (#97141).

Techniques: Expressing, Over Expression, Labeling

Journal: bioRxiv

Article Title: Inducible overexpression of zebrafish microRNA-722 suppresses chemotaxis of human neutrophil like cells

doi: 10.1101/578583

Figure Lengend Snippet: (A) Relative abundance of mRNA levels of indicated genes in vector or miR-722 expressing dHL-60 ± DOX. Results are presented as mean ± s.d. from three independent experiments. ****, p < 0.0001, Sidak’s multiple comparisons test. (B) Immunoblot of RAC2 in vector or miR-722 expressing dHL-60 cells ± DOX. Vinculin is used as a loading control. The blot was cut in half and probed with two different primary antibodies. Cropped images from the full length blot (Supplementary Figure 2) are shown. (C) Alignment of miR-722 seed sequence and the 7mer-m8 seed binding site in the RAC 2 3′UTR which is then mutated for the reporter assay in (E). (D) Schematics of the constructs used for the luciferase reporter assay. Wild-type or mutated RAC2 3′UTR were cloned downstream of a renilla luciferase gene. A firefly luciferase gene on the same plasmid was used as a normalization control. mir-722 or vector hairpin was cloned into a mammalian expression plasmid for expression. (E) Selective suppression of renilla luciferase activity by miR-722 through binding to the seed sequence in RAC 2 3′UTR. Results are presented as means ± s.d. from three independent experiments. ****, p < 0.0001, Mann–Whitney test.

Article Snippet: Plasmids are available on Addgene: pCDNA miR-722-Dendra (#97163), pcDNA3.1 Dendra2 (#103967), pSi-check2-hRac2 3′UTR (#97160), pSi-check2-hRac2 -mut 3′UTR (#97161), plix4.03-miR-722 (#97140), plix4.03-vector (#97141).

Techniques: Plasmid Preparation, Expressing, Western Blot, Sequencing, Binding Assay, Reporter Assay, Construct, Luciferase, Clone Assay, Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Inducible overexpression of zebrafish microRNA-722 suppresses chemotaxis of human neutrophil like cells

doi: 10.1101/578583

Figure Lengend Snippet: (A) Representative images and tracks of dHL-60 cells expressing either vector (left) or miR-722 with (right) and without (middle) doxycycline toward fMLP. Scale bar: 200 µm. (B, C) Quantification of velocity (B) and directionality (C) of dHL-60 cells during chemotaxis. Results are representative of three independent experiments. ****, p < 0.0001, Sidak’s multiple comparisons test. (D) Transwell migration of vector or miR-722 expressing dHL-60 cells towards fMLP. Results are average of three independent experiments and presented as mean ± s.d.. **, p < 0.01; ***, p<0.001, Sidak’s multiple comparisons test.

Article Snippet: Plasmids are available on Addgene: pCDNA miR-722-Dendra (#97163), pcDNA3.1 Dendra2 (#103967), pSi-check2-hRac2 3′UTR (#97160), pSi-check2-hRac2 -mut 3′UTR (#97161), plix4.03-miR-722 (#97140), plix4.03-vector (#97141).

Techniques: Expressing, Plasmid Preparation, Chemotaxis Assay, Migration