Journal: ACS Nano
Article Title: Caveolar Endocytosis Governs Nanoneedle Transfection
doi: 10.1021/acsnano.5c11011
Figure Lengend Snippet: Caveolin 1 knockout abolishes nanoneedle transfection in MG63 cells. (a) Western blot of CAV-1 expression in MG63 knockout cell clones (MG63 −ve C1, C2, and C3) and MG63 (MG63 +ve). (b–d) Representative confocal fluorescence image of CAV-1 expression and localization in (b) MG63 +ve, (c) MG63 −ve, and (d) Treg −ve cells after 6 h incubation. Scale bar: 2 μm. (e) CAV-1 expression levels in MG63 +ve, MG63 −ve, and Tregs. One-way ANOVA followed by Tukey’s multiple comparisons test. p -values are indicated above the bars. (f) Representative flow cytometry histograms of CTxB uptake in MG63 +ve and MG63 −ve for Flat and nN. (g) Cholera toxin B (CTxB) delivery efficiency in MG63 +ve and MG63 −ve for Flat and nN. Data presented as mean ± SD, n = 3 independent samples, two-way ANOVA followed by Tukey’s multiple comparisons test. p -values are indicated above the bars. (h) Representative flow cytometry histograms of transferrin (Tfn) uptake in MG63 +ve and MG63 −ve for Flat and nN. (i) Tfn delivery efficiency in MG63 +ve and MG63 −ve for Flat and nN. Data presented as mean ± SD, n = 3 independent samples, two-way ANOVA followed by Tukey’s multiple comparisons test. p -values are indicated above the bars. (j) Representative flow cytometry contour plots of the delivery ( Y -axis, Cy5 fluorescence) and transfection ( X -axis, eGFP fluorescence) of Cy5-eGFP in MG63 +ve and MG63 −ve for Flat and nN. (k) Delivery efficiency (Cy5 + cell population: Q1 + Q2) in MG63 +ve and MG63 −ve for Flat and nN. Data presented as mean ± SD, n = 3 independent samples, two-way ANOVA followed by Tukey’s multiple comparisons test. p -values are indicated above the bars. (l) MFI values of Cy5 for groups presented in (k). Data presented as mean ± SD, n = 3 independent samples, two-way ANOVA followed by Tukey’s multiple comparisons test. p -values are indicated above the bars. (m) Transfection efficiency (Cy5 + eGFP + cell population: Q2) in MG63 +ve and MG63 −ve for Flat and nN. Data presented as mean ± SD, n = 3 independent samples, two-way ANOVA, followed by Tukey’s multiple comparisons test. p -values are indicated above the bars. (n) MFI values of eGFP fluorescence for groups presented in (m). Data presented as mean ± SD, n = 3 independent samples, two-way ANOVA, followed by Tukey’s multiple comparisons test. p -values are indicated above the bars.
Article Snippet: The CAV-1 sgRNA target sequence (AUGUUGCCCUGUUCCCGGAU) was ordered from Synthego.
Techniques: Knock-Out, Transfection, Western Blot, Expressing, Clone Assay, Fluorescence, Incubation, Flow Cytometry