Fig. 6: Augmin and γ-TuRC are stabilized in the interphase centriole by POC5-Augmin interaction and released upon mitotic onset.

a Representative immunofluorescence images of RPE1 cells in different cell cycle stages stained against γ-tubulin (yellow) and HAUS1-SNAP-S with anti-Strep antibodies (HAUS1-Strep, magenta). DNA was visualized with DAPI (blue). Green arrows indicate the centrioles shown in the close-up view. b, c Quantification of fluorescence intensity at centrosomes for the indicated proteins shown in a) and Supplementary Fig. 23. n = 125 (interphase), 132 (prophase), 121 (prometaphase), 125 (metaphase), 162 (anaphase), 128 (telophase), 126 (+ STLC), and 117 (+ STLC&BI2536) centrioles were analyzed for HAUS4 intensity. n = 108 (interphase), 127 (prophase), 121 (prometaphase), 126 (metaphase), 121 (anaphase), 122 (telophase), 120 (+ STLC), and 120 (+ STLC&BI2536) centrioles were analyzed for γ-tubulin intensity. n = 127 (interphase), 118 (prophase), 130 (prometaphase), 123 (metaphase), 121 (anaphase), 127 (telophase), 131 (+ STLC), and 119 (+ STLC&BI2536) centrioles were analyzed for POC5 intensity. d Immunoblot showing protein levels of HAUS4 and γ-tubulin in wild-type control, POC5^-/-, POC5 and POC5^mut cells. e Quantification of relative HAUS4 and γ-tubulin levels in (d) (normalized to actin). f Representative immunoblots of POC5^-/- cells expressing POC5 or POC5^mut, treated with 50 µg/ml CHX for the indicated durations, using anti-HAUS4, anti-γ-tubulin and anti-GAPDH (loading control) antibodies. g) Quantification of panel f). Relative protein levels were normalized to protein levels at time 0. Linear regression was performed; R² values are provided in the Source Data. h Representative images of wild-type control, POC5^-/-, POC5, and POC5^mut cells in prophase and metaphase stained against the centrosomal marker γ-tubulin (green). DNA was visualized with DAPI. Green arrows indicate misaligned chromosomes. i Quantification of cells with amplified γ-tubulin foci and misaligned chromosomes for h). N = 139 (control), 136 (POC5^-/-), 129 (POC5), and 137 (POC5^mut) cells were analyzed for amplified γ-tubulin foci. n = 135 (control), 133 (POC5^-/-), 125 (POC5) and 131 (POC5^mut) cells were analyzed for misaligned chromosomes. (a), (h), scale bars: 10 µm, magnification scale bars: 1 µm; in (b, c, e, i), data are presented as mean ± SD, and all statistics were derived from ordinary one-way ANOVA analysis of N = 3 biologically independent experiments. Source data are provided as a Source Data file.