Journal: bioRxiv
Article Title: BMI-1 Modulation and Trafficking During M Phase in Diffuse Intrinsic Pontine Glioma
doi: 10.1101/2025.05.16.654605
Figure Lengend Snippet: (A) Scheme for the cell synchronization studies (top) and corresponding cell cycle analysis by flow cytometry (bottom). The percentage of cells in each cell cycle is indicated. (B) Immunoblot analysis of BMI-1 at the indicated timepoints. The arrow indicates phosphorylated BMI-1. β-Actin is used as the loading control. (C) Immunoblot analysis of BMI-1, Cyclin B1 and histone H3 S10-P in the cytoplasmic (C) and nuclear (N) fractions when treated with PTC596 (100 nM for 24 h) or colchicine (100 ng/ml for 24 h). β-Actin and total H3 served as loading controls. Arrow indicates phosphorylated BMI-1. (D) Representative immunofluorescence images showing DAPI (blue), Cyclin B1 (green) and BMI-1 (red) in cells treated with PTC596 or colchicine at the indicated doses for 24 h. White arrows indicate the cells with cytoplasmic localization of BMI-1. (E) immunoblot analysis of RING1B in the cytoplasmic (C) and nuclear (N) fractions in cells treated with PTC596 (100 nM for 24h) or colchicine (100 ng/ml for 24h). (F) Representative immunofluorescence images showing DAPI (blue), H3-S10-P (green) and RING1B (red) in cells treated with 100 nM PTC596 for 24 h. White arrows indicate the cells with cytoplasmic localization of BMI-1. (G) Representative immunofluorescence images showing DAPI (blue), H3-S10-P (green) and RING1B (red) in cells treated with 100 nM PTC596 for 24 h. Yellow arrows indicate the cells with RING1B still localized within DNA. All experiments were conducted in CCHMC-DIPG-1 cells at n=2. P values are indicated (*, P < 0.05; ns, not significant (P>0.05))
Article Snippet: Primary antibodies were used against BMI-1 (Cell signaling, Cat# 6964, RRID: AB_10828713, 1:500), RING1B (Cell signaling, Cat# 5694, RRID: AB_10705604, 1:500), H3 S10-P (Cell signaling, Cat# 9706, RRID: AB_331748, 1:500), Cyclin B1 (Cell signaling, Cat# 4138, RRID: AB_2072132, 1:500) were stained with corresponding secondary antibodies, Alexa Fluor® 488 AffiniPureTM Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, Cat# 711-545-152, RRID: AB_2313584, 1:500) or Alexa Fluor® 594 AffiniPureTM Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, Cat# 715-585-150, RRID: AB_2340854, 1:500).
Techniques: Cell Cycle Assay, Flow Cytometry, Western Blot, Control, Immunofluorescence
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