Analysis of HN1 and HN1L double knock-out cells. (A) Table showing the primary and secondary screen CRISPR score of HN1 or HN1L iKOs in K562 cells. (B) Representative confocal immunofluorescence images of mitotic metaphase and interphase HeLa cells showing the localization of GFP-tagged HN1 and HN1L proteins, Scale bar: 5 µm. (C) Percentage of mitotic cells with mitotic defects in control and after HN1+HN1L double iKO, treated with nocodazole (noc), paclitaxel (pac), or STLC. n > 100 cells per condition, across three experimental replicates. (D) Representative Z-projected deconvolved immunofluorescence images of mitotic cells of HN1+HN1L double iKO in HeLas treated with nocodazole, paclitaxel, or STLC. Microtubules (DM1α), DNA (DAPI). Scale bar: 10 µm. (E) Left: Live confocal stills of td-Tomato EB3 expressing control or HN1+HN1L double iKO HeLa cells; middle: max projection over 30 s middle; right: representative kymographs generated from region highlighted by red arrows. Scale bars: 5 µm/2 µm (kymograph). (F) EB3 speed quantification in control, HN1, HN1L, or HN1/HN1L double iKO HeLa cells. n = 93, 103, 104, 127 kymographs, n > 31 cells across three experimental replicates. (G) Quantification of total spindle tubulin signal in the control or HN1+HN1L double iKO HeLa cells. n = 70, 59 across three experimental replicates. (H) Quantification of total EB1 immunofluorescence in control or HN1+HN1L double iKO HeLa cells. N = 76, 75 across three experimental replicates. Statistical tests performed: Welch’s t test (ns = not significant, ***P = <0.001, ****P = <0.0001). Blue lines indicate the median.