Figure 6. Super-resolution imaging of astral microtubules in DYNLL1-, CHICA-, and HMMR-depleted cells. (A) HeLa cells were transfected with control, HMMR, CHICA, or DYNLL1 siRNA duplexes for 72 h. The cells were fixed and then stained for CHICA, tubulin, and with DAPI to reveal DNA. Efficient depletion of CHICA and HMMR in these cells was confirmed by the loss of CHICA from the spindle microtubules (not depicted). Samples were analyzed by super-resolution imaging, and maximum intensity projections of the 3D-SIM stacks are shown from a viewpoint perpendicular to the pole-to-pole axis of the spindle. Bar, 5 µm. (B) To test the importance of astral microtubules in spindle positioning and rotation, HeLa cells were treated with a subcritical dose of nocodazole (6.25 ng/ml) for 10 min, fixed, and then stained for tubulin, CHICA, and with DAPI to reveal DNA. CHICA-C antibody was used for staining. Controls were left untreated. Example images are shown in the figure. Bar, 10 µm. (C) The number of cells with normally centered, rotated, or displaced spindles was counted and plotted in the graph. Error bars indicate the SEM (n = 50 in two independent experiments). (D) HeLa cells expressing mCherry-tagged EB1 were transfected with control, HMMR, CHICA, or DYNLL1 siRNA duplexes for 72 h, or CenpE siRNA duplexes for 48 h. Cells were imaged at four planes positioned at the cell equator to cut through one or both spindle poles. Acquisition was with 30% laser power, 100-ms exposure time at maximum speed, equivalent to 0.87 frames per second. EB1 comets located between the spindle poles and the cell cortex, which mark growing astral microtubule ends, were identified and marked by eye in ImageJ and the mean distance moved per unit time calculated and plotted in the bar graph. Error bars indicate the SEM (n = 20).
Figure 6.

Super-resolution imaging of astral microtubules in DYNLL1-, CHICA-, and HMMR-depleted cells. (A) HeLa cells were transfected with control, HMMR, CHICA, or DYNLL1 siRNA duplexes for 72 h. The cells were fixed and then stained for CHICA, tubulin, and with DAPI to reveal DNA. Efficient depletion of CHICA and HMMR in these cells was confirmed by the loss of CHICA from the spindle microtubules (not depicted). Samples were analyzed by super-resolution imaging, and maximum intensity projections of the 3D-SIM stacks are shown from a viewpoint perpendicular to the pole-to-pole axis of the spindle. Bar, 5 µm. (B) To test the importance of astral microtubules in spindle positioning and rotation, HeLa cells were treated with a subcritical dose of nocodazole (6.25 ng/ml) for 10 min, fixed, and then stained for tubulin, CHICA, and with DAPI to reveal DNA. CHICA-C antibody was used for staining. Controls were left untreated. Example images are shown in the figure. Bar, 10 µm. (C) The number of cells with normally centered, rotated, or displaced spindles was counted and plotted in the graph. Error bars indicate the SEM (n = 50 in two independent experiments). (D) HeLa cells expressing mCherry-tagged EB1 were transfected with control, HMMR, CHICA, or DYNLL1 siRNA duplexes for 72 h, or CenpE siRNA duplexes for 48 h. Cells were imaged at four planes positioned at the cell equator to cut through one or both spindle poles. Acquisition was with 30% laser power, 100-ms exposure time at maximum speed, equivalent to 0.87 frames per second. EB1 comets located between the spindle poles and the cell cortex, which mark growing astral microtubule ends, were identified and marked by eye in ImageJ and the mean distance moved per unit time calculated and plotted in the bar graph. Error bars indicate the SEM (n = 20).

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