Journal: EMBO Reports

Article Title: Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

doi: 10.15252/embr.202154160

Figure Lengend Snippet: A Total lysates from hTERT‐RPE1 cells were IP with anti‐myosin VI antibody and an anti‐rabbit IgG antibody (as control). IB was performed with anti‐OFD1 and anti‐myosin VI antibodies. B A scheme of the IF analysis performed to calculate the total intensity of OFD1 staining at the mother and daughter centrioles. C, D IF analysis of centriole‐associated OFD1 signal. hTERT‐RPE1 cells were transfected with siRNA against myosin VI. Four days after transfection, cells were treated with nocodazole (1 h, 6 μg/ml) and immunostained with anti‐OFD1, anti‐centrin1 and anti‐Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1‐only stained. A scheme of the position of the markers used is depicted above. (C) Representative images, scale bar, 1 μm. (D) Quantification of OFD1 intensity at the mother or daughter centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mother centrioles: Mock, n = 148 cells; MyoVI KD, n = 199 cells, from four independent experiments. Daughter centrioles: Mock, n = 101 cells; MyoVI KD, n = 148 cells, from three independent experiments. **** P < 0.0001 by Mann–Whitney test. E IB analysis of hTERT‐RPE cells treated with the indicated siRNAs with anti‐myosin VI and anti‐OFD1 antibodies. Anti‐GAPDH was used as loading control. F, G IF analysis of Cep164 signal. hTERT‐RPE1 cells were transfected with siRNA against myosin VI and/or OFD1. Four days after transfection, cells were treated with nocodazole (1 h, 6 μg/ml) and immunostained with anti‐OFD1, anti‐centrin1 and anti‐Cep164 antibodies. (F) Representative images. Scale bar, 1 μm. (G) Quantification of Cep164 intensity at the mother centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mock, n = 147 cells; OFD1 KD, n = 146 cells; MyoVI KD, n = 150 cells; MyoVI + OFD1 KD, n = 151 cells, from four independent experiments. ** P < 0.005; **** P < 0.0001 by Kruskal–Wallis test. Source data are available online for this figure.

Article Snippet: Cep164 , Goat , Santa‐Cruz Biotechnology , sc‐240226 , IF 1:700.

Techniques: Control, Staining, Transfection, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

doi: 10.15252/embr.202154160

Figure Lengend Snippet: Growth curve of hTERT‐RPE1 cells transfected with myosin VI siRNA. A representative plot of three independent experiments is shown. Growth curve of BJ‐hTERT cells transfected with myosin VI siRNA. A representative plot of two independent experiments is shown. Analysis of DNA content in hTERT‐RPE1 cells stably expressing a myosin VI shRNA. After 10 days of doxycycline induction, the cells were stained with propidium iodide (PI) and analysed by FACS. Ctrl, control cells, not induced. Senescence‐associated β‐gal assay (SA‐β‐gal) of cells treated as in C. Scale bar, 100 μm. IB analysis of hTERT‐RPE1 cells transfected with myosin VI siRNA, with anti‐myosin VI, anti‐p53 and anti‐p21 antibodies. Anti‐GAPDH was used as loading control. Growth curve of hTERT‐RPE1 cells transfected with the indicated p53 and myosin VI siRNAs. A representative plot of three independent experiments is shown. Representative bright‐field images of cells treated with the indicated p53 and myosin VI siRNAs. Scale bar, 200 μm. IB analysis with anti‐myosin VI, anti‐p53 and anti‐p21 antibodies of hTERT‐RPE cells treated with Nutlin‐3, or not treated as control. Anti‐GAPDH was used as loading control. Quantification of OFD1 intensity at the mother or daughter centrioles. hTERT‐RPE1 cells treated or not with Nutlin‐3 were incubated with nocodazole for 1 h (6 μg/ml) and immunostained with anti‐OFD1, anti‐centrin1 and anti‐Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1‐only stained. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mother centrioles: Mock, n = 148 cells; Nutlin‐3, n = 169 cells, from four independent experiments. Daughter centrioles: Mock, n = 101 cells; Nutlin‐3, n = 122 cells, from three independent experiments. ns, not significant by Mann–Whitney test. Quantification of Cep164 intensity at the mother centrioles in hTERT‐RPE1 cells treated as in (I). Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mock, n = 195 cells; Nutlin‐3, n = 196 cells, from four independent experiments. ns, not significant by Mann–Whitney test. Source data are available online for this figure.

Article Snippet: Cep164 , Goat , Santa‐Cruz Biotechnology , sc‐240226 , IF 1:700.

Techniques: Transfection, Stable Transfection, Expressing, shRNA, Staining, Control, Incubation, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

doi: 10.15252/embr.202154160

Figure Lengend Snippet: A Super‐resolution analysis of OFD1 localisation at the centrioles. hTERT‐RPE1 cells were transfected with siRNA against myosin VI, immunostained with anti‐OFD1 and anti‐acetylated tubulin antibodies and visualised using structured illumination microscopy (SIM). Representative images are shown, scale bar, 500 nm. A scheme of the estimated localisation of OFD1 in the two conditions is depicted on the right side. B Two‐colour SIM images in control and myosin VI‐depleted cells illustrating the localisation and distribution of the distal appendage markers Cep164 and ODF2 at the centrioles, stained with anti‐acetylated tubulin antibody. Representative images are shown; scale bar, 500 nm. C dSTORM super‐resolution analysis of the distal appendage markers FBF1 and ODF2 in control and myosin VI‐depleted cells. To emphasise the symmetry of the structures, the signals from all nine appendages were averaged (bottom). Representative images are shown; scale bar, 200 nm. D–F FRAP analysis of centriole‐associated GFP‐OFD1. hTERT‐RPE cells stably expressing GFP‐OFD1 and centrin1‐dTomato were transfected with siRNA against myosin VI. After four days, cells were treated with nocodazole (1 h, 6 μg/ml) and subjected to live‐cell imaging. (D) Left: a scheme of the localisation of GFP‐OFD1 and the centriole marker centrin1‐dTomato. Right: a scheme of photobleaching and recovery of GFP‐OFD1 at the centrioles. (E) A representative graph of one out of three experiments. For each time point, the fraction of recovery of GFP‐OFD1 is shown. Results are expressed as means with 95% confidence interval. n = 12 cells (Mock), n = 13 cells (MyoVI KD). (F) Quantification of the half‐time of fluorescence recovery (t 1/2 ,) and of the mobile fraction of GFP‐OFD1. Results are expressed as mean ± SD. n = 41 cells (Mock), n = 31 cells (MyoVI KD), from three independent experiments. ns, not significant; *** P < 0.0005 by unpaired t ‐test. Source data are available online for this figure.

Article Snippet: Cep164 , Goat , Santa‐Cruz Biotechnology , sc‐240226 , IF 1:700.

Techniques: Transfection, Microscopy, Control, Staining, Stable Transfection, Expressing, Live Cell Imaging, Marker, Fluorescence

Journal: EMBO Reports

Article Title: Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

doi: 10.15252/embr.202154160

Figure Lengend Snippet: A scheme of the IF analysis performed to calculate the total intensity of satellite staining that surrounds the centrioles. The centriole marker Cep135 or Cep164 are used to define the centre of a 3 μm circle, in which the intensity of the satellite marker PCM1 was calculated. IF analysis of PCM1 signal. hTERT‐RPE1 cells were transfected with siRNA against myosin VI and immunostained with anti‐PCM1 and anti‐Cep135 antibodies. Upper panel, representative images, scale bar, 2 μm. Lower panel, quantification of PCM1 intensity. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mock, n = 96 cell; MyoVI KD, n = 98 cells, from two independent experiments. **** P < 0.0001 by Mann–Whitney test. IF analysis of PCM1 signal. hTERT‐RPE1 cells were treated with Nutlin‐3 for 24 h and immunostained with anti‐PCM1 and anti‐Cep135 antibodies. Panels as in B. Mock, n = 96 cells; Nutlin‐3, n = 100 cells, from two independent experiments. **** P < 0.0001 by Mann–Whitney test. IF analysis of PCM1 signal. hTERT‐RPE1 p53 KO cells were transfected with siRNA against myosin VI and immunostained with anti‐PCM1 and anti‐Cep164 antibodies. Panels as in (B). Quantification of PCM1 intensity refers to a 3 μm circle around the mother centriole, identified with anti‐Cep164 staining. Mock, n = 128 cells; MyoVI KD, n = 114 cells from three independent experiments. ns, not significant by Mann–Whitney test. Source data are available online for this figure.

Article Snippet: Cep164 , Goat , Santa‐Cruz Biotechnology , sc‐240226 , IF 1:700.

Techniques: Staining, Marker, Transfection, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

doi: 10.15252/embr.202154160

Figure Lengend Snippet:

Article Snippet: Cep164 , Goat , Santa‐Cruz Biotechnology , sc‐240226 , IF 1:700.

Techniques: Generated, Purification

Journal: Cell Communication and Signaling : CCS

Article Title: Tau-tubulin kinase 2 restrains microtubule-depolymerizer KIF2A to support primary cilia growth

doi: 10.1186/s12964-025-02072-8

Figure Lengend Snippet: Truncated TTBK2 proteins display reduced biochemical activity (1A) study outline; (1B) TTBK2 constructs used in this study; (1C-D) Western blot analysis of lysates from HEK293T cells transfected with Myc-CEP164 NT and Flag-TTBK2 constructs; (1E-F) hTERT RPE-1 TTBK2 KO cells DOX-inducibly expressing Flag-TTBK2 constructs were fixed and stained for pCEP164 (scale bars: 0.5μm); (1F) The intensity of pCEP164 centriolar signal was quantified from the images (4 independent experiments, n≥79 cells per condition, normalized to gTUB, one-way ANOVA, ****P<0.0001); (1G-I) Western blot analysis of lysates from HEK293T cells transfected with DVL3 and Flag-TTBK2 constructs

Article Snippet: Antibody against pS201 in CEP164 was custom-derived by Moravian Biotechnology Ltd ( https://www.moravian-biotech.com ).

Techniques: Activity Assay, Construct, Western Blot, Transfection, Expressing, Staining