Figure S2.
mDHC-GFP is functional in HeLa cells depleted of endogenous DHC. (a) Spinning disk microscopy images of live cells expressing Gal4T mCherry (magenta) and mDHC-GFP (green). The cells were treated with 25 nM siRNA against endogenous hDHC. The cell on the left had little mDHC-GFP signal and a dispersed Golgi apparatus whereas the cell on the right expressed higher levels of mDHC-GFP and had the Golgi clustered at the cell center. Quantification revealed that 100% of the cells without mDHC-GFP expression had a dispersed Golgi whereas only 24% of the cells expressing mDHC-GFP had a dispersed Golgi, indicating mDHC-GFP was functional (n = 34 cells from n = 2 independent experiments). The mean pixel intensities (in arbitrary units [au]) of the Gal4T and mDHC-GFP channels are indicated below the images in blue. (b) Representative western blot to verify the knock down of hDHC by RNAi in a. Quantification of the western blot confirmed that the RNAi successfully knocked down levels of hDHC by >90% (n = 2 independent experiments). (c) HILO microscopy image (left) from a 10-s-long time-lapse of mDHC-GFP in cells treated with 25 nM NC siRNA and the corresponding kymograph (right). Representative stationary, minus end–directed, and plus end–directed events are indicated with white, teal, and magenta arrowheads, respectively, in the kymograph and in the insets below. (d) HILO microscopy image (left) from a 10-s-long time-lapse of mDHC-GFP in cells treated with 25 nM hDHC siRNA and the corresponding kymograph (right). Representative stationary, minus end–directed, and plus end–directed events are indicated with white, teal, and magenta arrowheads, respectively, in the kymograph and in the insets below. (e) Representative western blot to verify the knockdown of DHC by RNAi in d. Quantification of the western blot confirmed that the RNAi successfully knocked down levels of DHC by an average of 72% (n = 2 independent experiments). (f) Comparison of displacement versus time plots of mDHC-GFP molecules in cells treated with 25 nM NC siRNA (left) and 25 nM siRNA against endogenous hDHC (right). In cells treated with 25 nM NC siRNA, 53% of the molecules remained stationary, 31% moved toward the minus ends of the MTs, and 16% moved toward the plus ends of the MTs, whereas in cells treated with 25 nM siRNA against hDHC, 52% of the molecules remained stationary, 29% moved toward the minus ends of the MTs, and 19% moved toward the plus ends of the MTs. (g) Box plots comparing the residence time of mDHC-GFP molecules in cells treated with 25 nM NC siRNA versus cells treated with 25 nM siRNA against endogenous hDHC indicating no significant differences. (h) Comparison of duration of plus end– and minus end–directed runs of mDHC-GFP in cells with NC RNAi and hDHC RNAi. “ns” represents no significant difference (Mann–Whitney U test). (i) Box plots comparing minus end–directed velocities (teal boxes) of mDHC-GFP molecules in cells treated with 25 nM NC siRNA and 25 nM siRNA against endogenous DHC, showing no significant differences. Similarly, the plus end–directed velocities (magenta boxes) were also not significantly different. The mean ± SD of velocities is indicated in the box plot. In f–i, data for NC was obtained from n = 254 binding events tracked from n = 3 independent experiments with >30 cells. Data for RNAi was obtained from n = 245 binding events tracked from n = 3 independent experiments with >30 cells. (j) Immunofluorescence images of MTs (top left, magenta), p150 (bottom left, green), and their merge (top right), obtained using Airyscan confocal microscopy showing enrichment of p150 at MT tips. The inset indicated with the white box is on the bottom right, with the normalized intensity profile of p150 along the MT indicated with the dashed white line, showing localization of p150 along the entire MT. Compare this image to Fig. 2 a, imaged with another p150 antibody. (k) Fluorescence images of anti-EB1 (left, green), anti-DYNC1H1 (magenta, center), and their merge (right) obtained using Airyscan confocal microscopy. (l) Fluorescence images of anti-EB1 (left, green), EB1-GFP (top center, magenta), anti-Rab5 (bottom center, magenta), and their merge (right), obtained using Airyscan confocal microscopy. (m) Quantification (mean ± SD) of the co-occurrence of the different proteins imaged in k and l. Note that “EB1 with EB1-GFP” is a positive control and represents the maximum co-occurrence that is quantifiable in cells; “Rab5 with EB1” is a negative control, since EB1 and Rab5 do not typically interact. Each dot represents an individual cell analyzed (n = 13–74 cells from n = 2–3 independent experiments); error bars represent SD. In c and d, “N” marks the location/direction of the nucleus. Source data are available for this figure: SourceData FS2.

mDHC-GFP is functional in HeLa cells depleted of endogenous DHC. (a) Spinning disk microscopy images of live cells expressing Gal4T mCherry (magenta) and mDHC-GFP (green). The cells were treated with 25 nM siRNA against endogenous hDHC. The cell on the left had little mDHC-GFP signal and a dispersed Golgi apparatus whereas the cell on the right expressed higher levels of mDHC-GFP and had the Golgi clustered at the cell center. Quantification revealed that 100% of the cells without mDHC-GFP expression had a dispersed Golgi whereas only 24% of the cells expressing mDHC-GFP had a dispersed Golgi, indicating mDHC-GFP was functional (n = 34 cells from n = 2 independent experiments). The mean pixel intensities (in arbitrary units [au]) of the Gal4T and mDHC-GFP channels are indicated below the images in blue. (b) Representative western blot to verify the knock down of hDHC by RNAi in a. Quantification of the western blot confirmed that the RNAi successfully knocked down levels of hDHC by >90% (n = 2 independent experiments). (c) HILO microscopy image (left) from a 10-s-long time-lapse of mDHC-GFP in cells treated with 25 nM NC siRNA and the corresponding kymograph (right). Representative stationary, minus end–directed, and plus end–directed events are indicated with white, teal, and magenta arrowheads, respectively, in the kymograph and in the insets below. (d) HILO microscopy image (left) from a 10-s-long time-lapse of mDHC-GFP in cells treated with 25 nM hDHC siRNA and the corresponding kymograph (right). Representative stationary, minus end–directed, and plus end–directed events are indicated with white, teal, and magenta arrowheads, respectively, in the kymograph and in the insets below. (e) Representative western blot to verify the knockdown of DHC by RNAi in d. Quantification of the western blot confirmed that the RNAi successfully knocked down levels of DHC by an average of 72% (n = 2 independent experiments). (f) Comparison of displacement versus time plots of mDHC-GFP molecules in cells treated with 25 nM NC siRNA (left) and 25 nM siRNA against endogenous hDHC (right). In cells treated with 25 nM NC siRNA, 53% of the molecules remained stationary, 31% moved toward the minus ends of the MTs, and 16% moved toward the plus ends of the MTs, whereas in cells treated with 25 nM siRNA against hDHC, 52% of the molecules remained stationary, 29% moved toward the minus ends of the MTs, and 19% moved toward the plus ends of the MTs. (g) Box plots comparing the residence time of mDHC-GFP molecules in cells treated with 25 nM NC siRNA versus cells treated with 25 nM siRNA against endogenous hDHC indicating no significant differences. (h) Comparison of duration of plus end– and minus end–directed runs of mDHC-GFP in cells with NC RNAi and hDHC RNAi. “ns” represents no significant difference (Mann–Whitney U test). (i) Box plots comparing minus end–directed velocities (teal boxes) of mDHC-GFP molecules in cells treated with 25 nM NC siRNA and 25 nM siRNA against endogenous DHC, showing no significant differences. Similarly, the plus end–directed velocities (magenta boxes) were also not significantly different. The mean ± SD of velocities is indicated in the box plot. In f–i, data for NC was obtained from n = 254 binding events tracked from n = 3 independent experiments with >30 cells. Data for RNAi was obtained from n = 245 binding events tracked from n = 3 independent experiments with >30 cells. (j) Immunofluorescence images of MTs (top left, magenta), p150 (bottom left, green), and their merge (top right), obtained using Airyscan confocal microscopy showing enrichment of p150 at MT tips. The inset indicated with the white box is on the bottom right, with the normalized intensity profile of p150 along the MT indicated with the dashed white line, showing localization of p150 along the entire MT. Compare this image to Fig. 2 a, imaged with another p150 antibody. (k) Fluorescence images of anti-EB1 (left, green), anti-DYNC1H1 (magenta, center), and their merge (right) obtained using Airyscan confocal microscopy. (l) Fluorescence images of anti-EB1 (left, green), EB1-GFP (top center, magenta), anti-Rab5 (bottom center, magenta), and their merge (right), obtained using Airyscan confocal microscopy. (m) Quantification (mean ± SD) of the co-occurrence of the different proteins imaged in k and l. Note that “EB1 with EB1-GFP” is a positive control and represents the maximum co-occurrence that is quantifiable in cells; “Rab5 with EB1” is a negative control, since EB1 and Rab5 do not typically interact. Each dot represents an individual cell analyzed (n = 13–74 cells from n = 2–3 independent experiments); error bars represent SD. In c and d, “N” marks the location/direction of the nucleus. Source data are available for this figure: SourceData FS2.

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