Journal: Scientific Reports

Article Title: The autophagy inducer SMER28 attenuates microtubule dynamics mediating neuroprotection

doi: 10.1038/s41598-022-20563-3

Figure Lengend Snippet: SMER28 treatment stabilizes microtubules by α-tubulin acetylation. ( a ) Western Blot analysis of U-2 OS cells treated with 50 and 200 µM of SMER28, 150 nM epothilone B (EpoB), 150 nM paclitaxel (Pacl) or 300 nM rapamycin (Rapa), as indicated, for 4 h. Lysates were analyzed for the protein levels of acetylated (ac)-α-tubulin. GAPDH served as loading control. ( b ) Quantification of relative ratios of ac-α-tubulin to GAPDH as indicated. Data show means of protein levels derived from six independent experiments ± s.e.m. *P < 0.05, ***P < 0.001, Mann–Whitney rank sum test. ( c ) U-2 OS cells were treated with DMSO as vehicle control, 50 µM or 200 µM SMER28, 150 nM epothilone B, 150 nM paclitaxel, 300 nM rapamycin or HBSS, as indicated, for 4 h. Cells were stained for ac-α-tubulin and visualized by SIM. Images show maximum intensity projections. ( d ) 3D object measurement tool of NIS Elements (Nikon) was used to measure the ac-tubulin filament volume of U-2 OS cells treated as indicated. SIM image stacks were used for quantification. Data show means of ac-tubulin filament volume in µm 3 from at least ten cells ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0001, one way ANOVA.

Article Snippet: For SIM (3D structured illumination microscopy) images shown in Figs. and , an N-SIM E (Nikon) was used, built on a Ti-Eclipse microscope (Nikon).

Techniques: Western Blot, Derivative Assay, MANN-WHITNEY, Staining

Journal: Cell Death and Differentiation

Article Title: SUGT1 controls susceptibility to HIV-1 infection by stabilizing microtubule plus-ends

doi: 10.1038/s41418-020-0573-5

Figure Lengend Snippet: a Representative SIM micrograph of 4 h HIV-1ΔEnvNL4-3-GFP-Vpr (VSV-G)-infected U2OS cells showing SUGT1 and α-tubulin expression (aI). aII is a magnification of the dashed region in aI. aII1–4 are the magnifications of the dashed regions in aII. Fluorescence overlap spectrums of aII1–4 cropped regions, are shown. b HIV-1 IN and SUGT1 expression levels by WB in control and SUGT1-depleted U2OS cells after 48 h siRNA transfection and expression of exogenous HIV-1 IN for 24 h. Representative confocal micrographs of HIV-1 HA-IN expression in control and SUGT1-depleted U2OS cells (c) and percentages of cells showing nuclear or diffused HIV-1 IN (d). e Immunoprecipitation of HA-IN in control and HA-IN-overexpressing HEK293T cells and expression of indicated proteins by WB. WB and images are representative of three independent experiments. Means ± SEM are indicated from at least three independent experiments. P values were calculated using the two-way ANOVA test (****p < 0.0001).

Article Snippet: For U2OS cells, acquisitions were performed in 3D SIM mode, with a N-SIM structured illumination Nikon microscope before image reconstruction using the NIS Elements software.

Techniques: Infection, Expressing, Fluorescence, Transfection, Immunoprecipitation