Figure S3.
Analysis of identified hits 2. (A) Clustal analysis of HN1, HN1L homologs across species. (B) Quantification of total spindle tubulin immunofluorescence signal in control, HN1, or HN1L iKO HeLa cells. n = 64, 70, 62 across three experimental replicates. (C) EB3 speed quantification in control or HN1+HN1L double iKO HeLa cells treated with nocodazole (noc) or paclitaxel (pac). n = 171, 188, 170, 162 kymographs, n = 31, 33, 32, 33 cells across three experiments. (D) Quantification of total mitotic spindle tubulin signal in the control or HN1+HN1L double iKO HeLa cells treated with nocodazole. n = 71, 72 across three experiments. (E) Quantification of total spindle tubulin signal in the HN1+HN1L double iKO HeLa cells treated with paclitaxel. n = 71, 72 across three experiments. (F) Live confocal image of td-Tomato EB3 in HeLa cells expressing control or HN1+HN1L double iKO. Stills were max projected over 30 s. Scale bar: 2 µm (G) Quantification of EB3 speed of cells in F n = 60, 63 kymographs, n = 24, 25 cells across three experiments. (H) Western blot of control, CARMT1 knockout, or CARNMT1 knock-out K562 cells expressing hardened CARNMT1 rescue construct probed for CARNMT1 or β-actin. (I) Percentage of cells appearing as monopolar in control, CARNMT1 knockout, or CARNMT1 knock-out K562 cells expressing hardened CARNMT1 rescue construct after STLC treatment. n > 100 cells each in three experiments. (J) Immunofluorescence images of CARNMT1 knock-out K562 cells expressing hardened CARNMT1 rescue construct stained for CARNMT1, tubulin, and DNA. Scale bar: 5 µm. (K) Representative max intensity projection of undeconvolved immunofluorescence images of iKO K562 s for control or CARNMT1. Stained for microtubules or DNA. Quantified in Fig. 5 F. (L) Quantification of mitotic spindle tubulin signal in control or CARNMT1 iKO HeLas. n = 58, 64 across three experiments. (M) Quantification of total EB1 immunofluorescence in control or CARNMT1 iKO K562 s. n > 60 across three experimental replicates. (N) Interphase EB3 speed quantification in control or CARNMT1 iKO K562 s expressing td-Tomato EB3. n = 130, 111 kymographs, n = 34, 35 cells across three experiments. Statistical tests performed: Welch’s t test (**P = <0.01, ***P = <0.001, ****p = <0.0001). Blue lines indicate the median. Source data are available for this figure: SourceData FS3.

Analysis of identified hits 2. (A) Clustal analysis of HN1, HN1L homologs across species. (B) Quantification of total spindle tubulin immunofluorescence signal in control, HN1, or HN1L iKO HeLa cells. n = 64, 70, 62 across three experimental replicates. (C) EB3 speed quantification in control or HN1+HN1L double iKO HeLa cells treated with nocodazole (noc) or paclitaxel (pac). n = 171, 188, 170, 162 kymographs, n = 31, 33, 32, 33 cells across three experiments. (D) Quantification of total mitotic spindle tubulin signal in the control or HN1+HN1L double iKO HeLa cells treated with nocodazole. n = 71, 72 across three experiments. (E) Quantification of total spindle tubulin signal in the HN1+HN1L double iKO HeLa cells treated with paclitaxel. n = 71, 72 across three experiments. (F) Live confocal image of td-Tomato EB3 in HeLa cells expressing control or HN1+HN1L double iKO. Stills were max projected over 30 s. Scale bar: 2 µm (G) Quantification of EB3 speed of cells in F n = 60, 63 kymographs, n = 24, 25 cells across three experiments. (H) Western blot of control, CARMT1 knockout, or CARNMT1 knock-out K562 cells expressing hardened CARNMT1 rescue construct probed for CARNMT1 or β-actin. (I) Percentage of cells appearing as monopolar in control, CARNMT1 knockout, or CARNMT1 knock-out K562 cells expressing hardened CARNMT1 rescue construct after STLC treatment. n > 100 cells each in three experiments. (J) Immunofluorescence images of CARNMT1 knock-out K562 cells expressing hardened CARNMT1 rescue construct stained for CARNMT1, tubulin, and DNA. Scale bar: 5 µm. (K) Representative max intensity projection of undeconvolved immunofluorescence images of iKO K562 s for control or CARNMT1. Stained for microtubules or DNA. Quantified in Fig. 5 F. (L) Quantification of mitotic spindle tubulin signal in control or CARNMT1 iKO HeLas. n = 58, 64 across three experiments. (M) Quantification of total EB1 immunofluorescence in control or CARNMT1 iKO K562 s. n > 60 across three experimental replicates. (N) Interphase EB3 speed quantification in control or CARNMT1 iKO K562 s expressing td-Tomato EB3. n = 130, 111 kymographs, n = 34, 35 cells across three experiments. Statistical tests performed: Welch’s t test (**P = <0.01, ***P = <0.001, ****p = <0.0001). Blue lines indicate the median. Source data are available for this figure: SourceData FS3.

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