Relationship between cell cycle stages and dispersion/scattering caused by various forms of active RB. (A and B) DOX-inducible active RB system in RPE1 cells. Addition of DOX causes the simultaneous expression of shRB1, which targets the 3′ UTR of RB1 and depletes endogenous RB protein, and exogenous mutant RB alleles (unphosphorylated ΔCDK-RB or any of the 14 mP-RB forms; A) or WT RB (B). (C) Cell cycle profile for ΔCDK-RB and WT RPE cells serum starved for 3 d (WT Ser Stav). (D) Percentage of G1 cells after 72-h DOX induction of mP-RB forms and ΔCDK-RB. Bar color and pattern depict categorization of dispersion phenotype for mP-RB forms and ΔCDK-RB. Red, blue, and white bars represent high, medium, and low disperser for both euchromatin and heterochromatin regions. Interleaved bars indicate different classifications for heterochromatin and euchromatin dispersion/scatter phenotype. (E and F) Percentage of dispersed and compact heterochromatin (Het) foci for the mP-RB forms and ΔCDK-RB. Bar color and pattern show intensity of dispersion phenotype, which was classified based on mean skeleton dot lengths. (G and H) Percentage of scattered and compact euchromatin (Eu) foci for the mP-RB forms and ΔCDK-RB. Bar color and pattern show intensity of scatter phenotype, which was classified based on mean skeleton dot lengths. Quantitation of dot lengths was from two independent biological replicates, set up and performed on different days. Error bars are SEM. Numbers of foci quantified for each sample (n) are as follows: E and F, n = 55 (356), 61 (ΔCDK-RB), 74 (608), 48 (795), 75 (826), 50 (612), 76 (230), 75 (811), 104 (249), 60 (788), 63 (821), 56 (373), 93 (807), 85 (252), 207 (WT Ser Stav), 115 (780); G and H, n = 84 (356), 60 (ΔCDK-RB), 52 (788), 113 (811), 124 (230), 144 (249), 66 (373), 120 (795), 80 (252), 80 (608), 106 (821), 56 (612), 145 (780), 69 (807), 99 (826), 111 (WT Ser Stav).
Figure S2.

Relationship between cell cycle stages and dispersion/scattering caused by various forms of active RB. (A and B) DOX-inducible active RB system in RPE1 cells. Addition of DOX causes the simultaneous expression of shRB1, which targets the 3′ UTR of RB1 and depletes endogenous RB protein, and exogenous mutant RB alleles (unphosphorylated ΔCDK-RB or any of the 14 mP-RB forms; A) or WT RB (B). (C) Cell cycle profile for ΔCDK-RB and WT RPE cells serum starved for 3 d (WT Ser Stav). (D) Percentage of G1 cells after 72-h DOX induction of mP-RB forms and ΔCDK-RB. Bar color and pattern depict categorization of dispersion phenotype for mP-RB forms and ΔCDK-RB. Red, blue, and white bars represent high, medium, and low disperser for both euchromatin and heterochromatin regions. Interleaved bars indicate different classifications for heterochromatin and euchromatin dispersion/scatter phenotype. (E and F) Percentage of dispersed and compact heterochromatin (Het) foci for the mP-RB forms and ΔCDK-RB. Bar color and pattern show intensity of dispersion phenotype, which was classified based on mean skeleton dot lengths. (G and H) Percentage of scattered and compact euchromatin (Eu) foci for the mP-RB forms and ΔCDK-RB. Bar color and pattern show intensity of scatter phenotype, which was classified based on mean skeleton dot lengths. Quantitation of dot lengths was from two independent biological replicates, set up and performed on different days. Error bars are SEM. Numbers of foci quantified for each sample (n) are as follows: E and F, n = 55 (356), 61 (ΔCDK-RB), 74 (608), 48 (795), 75 (826), 50 (612), 76 (230), 75 (811), 104 (249), 60 (788), 63 (821), 56 (373), 93 (807), 85 (252), 207 (WT Ser Stav), 115 (780); G and H, n = 84 (356), 60 (ΔCDK-RB), 52 (788), 113 (811), 124 (230), 144 (249), 66 (373), 120 (795), 80 (252), 80 (608), 106 (821), 56 (612), 145 (780), 69 (807), 99 (826), 111 (WT Ser Stav).

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