Figure S3.
Dynactin’s association with MTs. (a) Spinning disk microscopy images of live cells expressing mDHC-GFP, with no visible dynein clusters (left) and with distinct dynein clusters (yellow arrowheads) (right). (b) Box plots comparing the average intensities between cells with and without dynein clusters, showing that cells with higher total mDHC intensities exhibited clusters. Therefore, clusters of dynein were likely a result of high overexpression of mDHC. (n = 25 cells per condition from n = 1 independent experiment). (c) Left: Fluorescence images of p62-GFP (top, green), anti-p62 (magenta, middle), and their merge (bottom). Right: Fluorescence images of p150-GFP (green, top), anti-p150 (Invitrogen antibody, magenta, middle), and their merge (bottom). These images indicate that the antibodies employed to detect p62 and p150 in this study are specific. (d) Top: Immunofluorescence images of p62 (left, green), p150 (center, magenta), and their merge (right). Bottom: Immunofluorescence images of MT (left, cyan), EB1 (second from left, magenta), p150 (third from left, yellow), and their merge (right). All images were obtained using Airyscan confocal microscopy. (e) Quantification (mean ± SD) of the co-occurrence of the different proteins imaged in d. Note that “EB1 with EB1-GFP” has been reused from Fig. S2 k. Each dot represents an individual cell analyzed (n = 45–74 cells from n = 2–3 independent experiments). Error bars represent SD. All images were obtained using Airyscan confocal microscopy. (f) Immunofluorescence images of MT (magenta) and p150 (green) obtained using spinning disk microscopy + SRRF on cells treated with 35 nM NC siRNA (top) and 35 nM p150 siRNA (bottom). (g) Representative western blot to verify the knockdown of p150 by RNAi. Quantification of the western blot confirmed that the RNAi successfully knocked down levels of p150 by an average of 60% (two independent experiments). (h) Box plots comparing the mean intensity of p150 along MTs (representative ROI shown as a gray line in f) between cells treated with NC and p150 siRNA, showing that p150 knockdown reduces p150 levels along MTs. For each condition, 500 MT segments of length 15 ± 6 µm (mean ± SD) were analyzed from n = 50 cells from n = 2 independent experiments. (i) Immunofluorescence images of MT (magenta) and p62 (green) obtained using spinning disk microscopy + SRRF on cells treated with 35 nM NC siRNA (top) and 35 nM p150 siRNA (bottom). (j) Representative western blot to verify the knockdown of p150 by RNAi. Quantification of the western blot confirmed that the RNAi successfully knocked down levels of p150 by an average of 60% (n = 2 independent experiments). Moreover, there were no significant differences in the levels of p62 in cells treated with NC and p150 siRNA. (k) Box plots comparing the mean intensity of p62 along MTs (representative ROI shown as gray line in i) between cells treated with NC and p150 siRNA showing that p150 knockdown reduces p62 levels along MTs. For NC, ∼190 MT segments of length 15 ± 6 µm (mean ± SD) were analyzed from n = 51 cells from N = 2 independent experiments. For p150 RNAi, ∼100 MT segments of length 17 ± 9 µm (mean ± SD) were analyzed from n = 52 cells from n = 2 independent experiments.  (l) Representative western blot to verify the knockdown of hDHC by RNAi. Quantification of western blot confirmed that the RNAi successfully knocked down levels of endogenous hDHC by an average of 74% (n = 3 independent experiments). (m) Immunofluorescence images of p150 and MTs (left), and their merge (green and magenta, respectively, right) in NC (top) and hDHC siRNA cells (bottom), obtained using Airyscan confocal microscopy. (n) Quantification of the proportion of p150 that colocalized with MTs in NC and hDHC siRNA cells (n > 104 cells from n = 3 independent experiments, error bars represent SEM). In the NC cells, 63.2 ± 14.0% of p150 signal was on the MTs, whereas hDHC siRNA cells showed a small yet significant reduction (P < 0.01, Kruskal–Wallis test) to 57.3 ± 11.5% of p150 signal on MTs. This indicates that loss of DHC reduced p150’s loading to MTs only by a small extent of ∼6%. In a, f, and i, “N” marks the location/direction of the nucleus. Source data are available for this figure: SourceData FS3.

Dynactin’s association with MTs. (a) Spinning disk microscopy images of live cells expressing mDHC-GFP, with no visible dynein clusters (left) and with distinct dynein clusters (yellow arrowheads) (right). (b) Box plots comparing the average intensities between cells with and without dynein clusters, showing that cells with higher total mDHC intensities exhibited clusters. Therefore, clusters of dynein were likely a result of high overexpression of mDHC. (n = 25 cells per condition from n = 1 independent experiment). (c) Left: Fluorescence images of p62-GFP (top, green), anti-p62 (magenta, middle), and their merge (bottom). Right: Fluorescence images of p150-GFP (green, top), anti-p150 (Invitrogen antibody, magenta, middle), and their merge (bottom). These images indicate that the antibodies employed to detect p62 and p150 in this study are specific. (d) Top: Immunofluorescence images of p62 (left, green), p150 (center, magenta), and their merge (right). Bottom: Immunofluorescence images of MT (left, cyan), EB1 (second from left, magenta), p150 (third from left, yellow), and their merge (right). All images were obtained using Airyscan confocal microscopy. (e) Quantification (mean ± SD) of the co-occurrence of the different proteins imaged in d. Note that “EB1 with EB1-GFP” has been reused from Fig. S2 k. Each dot represents an individual cell analyzed (n = 45–74 cells from n = 2–3 independent experiments). Error bars represent SD. All images were obtained using Airyscan confocal microscopy. (f) Immunofluorescence images of MT (magenta) and p150 (green) obtained using spinning disk microscopy + SRRF on cells treated with 35 nM NC siRNA (top) and 35 nM p150 siRNA (bottom). (g) Representative western blot to verify the knockdown of p150 by RNAi. Quantification of the western blot confirmed that the RNAi successfully knocked down levels of p150 by an average of 60% (two independent experiments). (h) Box plots comparing the mean intensity of p150 along MTs (representative ROI shown as a gray line in f) between cells treated with NC and p150 siRNA, showing that p150 knockdown reduces p150 levels along MTs. For each condition, 500 MT segments of length 15 ± 6 µm (mean ± SD) were analyzed from n = 50 cells from n = 2 independent experiments. (i) Immunofluorescence images of MT (magenta) and p62 (green) obtained using spinning disk microscopy + SRRF on cells treated with 35 nM NC siRNA (top) and 35 nM p150 siRNA (bottom). (j) Representative western blot to verify the knockdown of p150 by RNAi. Quantification of the western blot confirmed that the RNAi successfully knocked down levels of p150 by an average of 60% (n = 2 independent experiments). Moreover, there were no significant differences in the levels of p62 in cells treated with NC and p150 siRNA. (k) Box plots comparing the mean intensity of p62 along MTs (representative ROI shown as gray line in i) between cells treated with NC and p150 siRNA showing that p150 knockdown reduces p62 levels along MTs. For NC, ∼190 MT segments of length 15 ± 6 µm (mean ± SD) were analyzed from n = 51 cells from N = 2 independent experiments. For p150 RNAi, ∼100 MT segments of length 17 ± 9 µm (mean ± SD) were analyzed from n = 52 cells from n = 2 independent experiments.  (l) Representative western blot to verify the knockdown of hDHC by RNAi. Quantification of western blot confirmed that the RNAi successfully knocked down levels of endogenous hDHC by an average of 74% (n = 3 independent experiments). (m) Immunofluorescence images of p150 and MTs (left), and their merge (green and magenta, respectively, right) in NC (top) and hDHC siRNA cells (bottom), obtained using Airyscan confocal microscopy. (n) Quantification of the proportion of p150 that colocalized with MTs in NC and hDHC siRNA cells (n > 104 cells from n = 3 independent experiments, error bars represent SEM). In the NC cells, 63.2 ± 14.0% of p150 signal was on the MTs, whereas hDHC siRNA cells showed a small yet significant reduction (P < 0.01, Kruskal–Wallis test) to 57.3 ± 11.5% of p150 signal on MTs. This indicates that loss of DHC reduced p150’s loading to MTs only by a small extent of ∼6%. In a, f, and i, “N” marks the location/direction of the nucleus. Source data are available for this figure: SourceData FS3.

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