1 / 4

Analysis of IFT88 Dynamics and Mitotic Index via siRNA in LLC-PK1 Cell Line

This study explores the role of IFT88 in mitosis using siRNA targeting IFT88 in LLC-PK1 cells. We present data on Western blotting (WB) to detect IFT88 and γ-tubulin, alongside imaging to quantify spindle defects and mitotic index. Supplementary figures illustrate changes in spindle morphology, phospho-Histone H3 positivity, and microtubule dynamics in response to IFT88 silencing. Key findings include alterations in spindle angle and increased spindle defects correlated with reduced IFT88 expression, offering insights into its role in mitotic fidelity and microtubule organization.

guy-pruitt
Télécharger la présentation

Analysis of IFT88 Dynamics and Mitotic Index via siRNA in LLC-PK1 Cell Line

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Supplementary Fig. S1 a. b. IFT88#2 IFT88 GFP siRNA: a tub/ IFT88 IFT88 100 75 WB anti-IFT88 50 WB anti-g tubulin c. siRNA: GFP 14 IFT88 10 Mitotic index (%) 6 2 T (h) 48 0 32 d. 20 0 -10 10 30 40 50 60 GFP siRNA: -10 0 40 160 220 280 300 330 IFT88 e. 60 siRNA: GFP IFT88 40 % cells 20 0 Time (min) 10-50 60-100 110-150 > 150

  2. Phos-H3 positive cells (Fold increase) 3 2 1 0 siRNA: MO: Con IFT88 control IFT88sc Supplementary Fig. S2 a. b. 90 siRNA: IFT88sc Control 70 72h 48h 72h 32h 48h 32h 100 50 % cells 75 30 WB anti-IFT88 75 10 Disrupted poles Disrupted g tubulin Misaligned chrom Spindle tilt WB anti-dynein IFT88sc siRNA: c. 40 control 30 IFT88sc 20 % cells w/ spindle defects GFP / a tubulin 10 0 GFP neg GFP pos GFP-IFT88 / LLC-PK1 d. a tubulin / g tubulin 100 Tg737 +/+ 80 Tg737 +/+ Tg737 -/- 60 % cells 40 20 0 Tg737 -/- 0 10 5 20 Spindle angle (a °) a tubulin/ g tubulin/ Phos-H3 e. Control MO IFT88 MO

  3. 16 12 g tubulin intensity (x105 a.u.) 8 4 0 siRNA: IFT88 GFP Supplementary Fig. S3 a. b. siRNA: IFT88 siRNA: GFP EB1 EB1 14000 14000 Gray scale 12000 Gray scale 12000 10000 10000 60000 60000 5051 5051 35000 35000 Gray scale Gray scale 10000 10000 0 10 20 0 10 20 Distance (mm) Distance (mm) c. d. IFT88/ EB1/ EB1 IFT88 DAPI 70 GFP 50 MT nucleation rate (min-1) siRNA: 30 10 EB1 siRNA: GFP IFT88

  4. Supplementary Fig. S4 a tub Dyn IC a tub/ Dyn a. a tub a tub/ Dyn Dyn IC a tub IgG a tub/ IgG a tub/ IgG IgG a tub Negative controls a tub/ a tub/ a tub No primary No primary a tub No primary No primary b. c. Mitotic shake off Mitotic shake off IP: Con Dyn IC Input IP: Con IFT88 Input 100 100 75 WB anti-IFT88 WB anti-IFT88 75 75 WB anti-Dynein IC WB anti-Dynein IC Mitotic shake off: Input IP Flag IP Ig d. IMCD IMCD Flag-IFT52 + - + - - - + - + + 100 IFT88 WB anti-IFT88 75 Flag-IFT52 WB anti-Flag 50 e. IP Ig IP IFT88 Input Mitotic shake off: IMCD IMCD Flag-IFT52 + - + - - + - + + - - + 100 IFT88 75 WB anti-IFT88 Flag-IFT52 50 WB anti-Flag

More Related