Figure 1.
Figure 1. pVHL attenuates MT growth rate. (A) RPE-1 cell system and depletion of pVHL using retroviral expression (pLMP-EB3GFP) of shRNAmir against VHL (shVHL) and a nonsilencing control (ns-control). (B) RCC-4 cell system lacking pVHL expression reconstituted with retroviral expression (pCMV(R)) of the wild-type long form pVHL30, the wild-type short form pVHL19, and the empty vector as control (VHL−/−). Vertical black lines indicate that intervening lanes have been spliced out. (A and B) Molecular mass is indicated in kilodaltons. (C) Immunofluorescence analysis of MT mass in RCC-4 cells expressing the wild-type long form pVHL30 or the empty vector (VHL−/−). ITub,rel indicates the α-tubulin signal intensity normalized to GSK-3β signal. (D) Relative α-tubulin signal as measured in C. Data were pooled from n = 3 slides, each slide with 10 stacks of five frames each per condition. Mean ± SD is shown (*, P < 0.02). (E) Single frame of a video acquired from RPE-1 cells expressing EB3-GFP. Red marks indicate automatic detection of EB3-GFP comets. (F) Mean nearest neighbor distance between EB3-GFP comets in RCC-4 cells (n = 15) expressing pVHL30 or the empty vector (VHL−/−). Box plot indicates distribution of n = 15 cells. (G) Overlay to frame in E of EB3-GFP tracks over 125 frames sampled at 0.5 s/frame. Track color encodes speed. (E and G) Zoom panels represent enlargements of the boxed areas in the left panels. (H) Distributions of growth speed of >55,000 tracked EB3-GFP comets in 12 RPE-1 cells (left) and of per cell means (right). Comparison of growth speed distributions was based on Kolmogorov-Smirnov test, and comparison of per cell means was based on the Student’s t test after validation of normality (***, P < 10−5; see Materials and methods). (I) Distributions of growth speed of all tracked EB3-GFP comets (left) and of per cell means (right) in RCC-4 cells (***, P < 10−5). See legend for H for statistical test procedures. (F, H, and I) Boxes indicate 25, 50 (median), and 75% quantiles; whiskers extend to 1.5× the interquartile range; red dots indicate outliers beyond this range. Notched boxes indicate uncertainty of the median. Boxes whose notches do not overlap indicate that the medians of the two clusters differ at the 5% significance level. Bars: (C) 10 µm; (E and G) 5 µm.

pVHL attenuates MT growth rate. (A) RPE-1 cell system and depletion of pVHL using retroviral expression (pLMP-EB3GFP) of shRNAmir against VHL (shVHL) and a nonsilencing control (ns-control). (B) RCC-4 cell system lacking pVHL expression reconstituted with retroviral expression (pCMV(R)) of the wild-type long form pVHL30, the wild-type short form pVHL19, and the empty vector as control (VHL−/−). Vertical black lines indicate that intervening lanes have been spliced out. (A and B) Molecular mass is indicated in kilodaltons. (C) Immunofluorescence analysis of MT mass in RCC-4 cells expressing the wild-type long form pVHL30 or the empty vector (VHL−/−). ITub,rel indicates the α-tubulin signal intensity normalized to GSK-3β signal. (D) Relative α-tubulin signal as measured in C. Data were pooled from n = 3 slides, each slide with 10 stacks of five frames each per condition. Mean ± SD is shown (*, P < 0.02). (E) Single frame of a video acquired from RPE-1 cells expressing EB3-GFP. Red marks indicate automatic detection of EB3-GFP comets. (F) Mean nearest neighbor distance between EB3-GFP comets in RCC-4 cells (n = 15) expressing pVHL30 or the empty vector (VHL−/−). Box plot indicates distribution of n = 15 cells. (G) Overlay to frame in E of EB3-GFP tracks over 125 frames sampled at 0.5 s/frame. Track color encodes speed. (E and G) Zoom panels represent enlargements of the boxed areas in the left panels. (H) Distributions of growth speed of >55,000 tracked EB3-GFP comets in 12 RPE-1 cells (left) and of per cell means (right). Comparison of growth speed distributions was based on Kolmogorov-Smirnov test, and comparison of per cell means was based on the Student’s t test after validation of normality (***, P < 10−5; see Materials and methods). (I) Distributions of growth speed of all tracked EB3-GFP comets (left) and of per cell means (right) in RCC-4 cells (***, P < 10−5). See legend for H for statistical test procedures. (F, H, and I) Boxes indicate 25, 50 (median), and 75% quantiles; whiskers extend to 1.5× the interquartile range; red dots indicate outliers beyond this range. Notched boxes indicate uncertainty of the median. Boxes whose notches do not overlap indicate that the medians of the two clusters differ at the 5% significance level. Bars: (C) 10 µm; (E and G) 5 µm.

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