pmcherry c1 mcherry nls  (Addgene inc)

Journal: bioRxiv

Article Title: A Myosin Nanomotor Essential for Stereocilia Maintenance Expands the Etiology of Hereditary Hearing Loss DFNB3

doi: 10.1101/2025.02.19.639121

Figure Lengend Snippet: MYO15A-3 traffics the elongation complex protein in filopodia. HeLa cells were transfected with plasmids encoding for protein fusion proteins, either EGFP-MYO15A-2 (positive control), EGFP-MYO15A-3 or EGFP-MYO10 (negative control), and co-transfected with either (A-C) mCherry-EPS8 or (H-J) mCherry-WHRN to detect endogenous protein. Cells were imaged using a spinning disk confocal microscope to investigate the localization and interaction of these proteins. The intensity of EGFP-Myosin and mCherry-EPS8/WHRN was measured blinded at multiple filopodia tips (∼300 filopodia from n=3 independent transfections). (D-F, K-M) The molecular interaction between the myosin and the EC proteins was determined using Pearson’s r coefficient, calculated along the filopodia shaft to measure the fluorescence correlation between the bait and prey. (G, N) Each data point represents the average interaction index from a single experiment (3 independent determinations) with a total of 30 cells per condition. The data are presented as mean ± SD with statistical significance determined using One-way ANOVA. *** P<0.001, **** P<0.0001. Scale bars: 5 µm.

Article Snippet: The pmCherry-C1-EPS8 plasmid was a gift from Christien Merrifield (Addgene plasmid #29779).

Techniques: Transfection, Positive Control, Negative Control, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: A Myosin Nanomotor Essential for Stereocilia Maintenance Expands the Etiology of Hereditary Hearing Loss DFNB3

doi: 10.1101/2025.02.19.639121

Figure Lengend Snippet: MYO15A-3 is not required for the elongation complex trafficking in hair cells at P4, but traffics the elongation complex at P21. (A, C) Confocal images of hair cells from cochleae of Myo15a (+/ Δ 3) and Myo15a ( Δ 3/ Δ 3) mice at postnatal day 4 (P4) immunolabeled for the EPS8/ WHRN (green). The hair cells were counterstained with phalloidin (magenta) to visualize the actin-rich stereocilia bundles. (B, D) Quantification of EPS8 and WHRN relative intensity at the stereocilia tips. Each dot represents the fluorescence intensity of a single hair cell. This was measured by averaging the absolute fluorescence intensity at the tips of individual row 1 stereocilia. Data are mean ± SD for n=3 mice per genotype (total ∼33-38 hair cells per condition/genotype). The absolute fluorescence for EPS8/WHRN was not significantly different between genotypes, as determined via the Mann-Whitney U-test to measure EPS8 level and an unpaired t-test for WHRN. (E, G) Confocal images of hair cells from cochleae of Myo15a (+/ Δ 3) and Myo15a ( Δ 3/ Δ 3) mice at postnatal day 21 (P21) immunolabeled for the EPS8/ WHRN (green). (F, H) Quantification of EPS8 and WHRN relative intensity at the stereocilia tips. Data are mean ± SD for n=3 mice per genotype (total ∼28-35 hair cells per condition/genotype) with statistical significance of **** P<0.0001 via Mann-Whitney U-test to measure WHRN and an unpaired t-test for EPS8. Scale bars: 5 µm.

Article Snippet: The pmCherry-C1-EPS8 plasmid was a gift from Christien Merrifield (Addgene plasmid #29779).

Techniques: Immunolabeling, Fluorescence, MANN-WHITNEY