Journal: Frontiers in Cell and Developmental Biology

Article Title: Involvement of RBP-J interacting and tubulin-associated protein in the distribution of protein regulator of cytokinesis 1 in mitotic spindles

doi: 10.3389/fcell.2024.1472340

Figure Lengend Snippet: Knockdown of RITA results in mitotic defects. (A) Cellular lysates from HeLa CRISPRi sgRITA and control cells were prepared for Western blot analysis with RITA antibody. GAPDH served as loading control. (B) The average protein levels of RITA normalized to the loading control in HeLa CRISPRi cells. The results are from three independent experiments. (C) Total RNAs were extracted for RT-PCR and gene levels of RITA are shown for HeLa CRISPRi sgcon and sgRITA cells. The results are from three individual experiments. (D) HeLa CRISPRi sgRITA cells and control HeLa CRISPRi sgcon cells were stained for the MT marker α-tubulin (green), the kinetochore/centromere marker ACA (yellow) and DNA (DAPI, blue) for microscopy. Representative confocal images are shown. White arrows indicate defects in chromosome alignment in metaphase and chromosome segregation in anaphase. Scale: 10 µm. (E, F) Chromosome congression (E) and segregation defects (F) were evaluated in HeLa CRISPRi sgRITA and HeLa CRISPRi sgcon cells. Three individual experiments were performed (n = 3, 100 mitotic cells for each condition in each experiment) and the results are shown as mean ± SEM. (G) HeLa cells were transiently transfected with siRNA #1 targeting the coding region of RITA or siRNA #2 targeting its 3′-untranslated region for 48 h and stained as indicated. Representative confocal images are shown. White arrows depict failures in chromosome congression and segregation. Scale: 10 µm. (H, I) Evaluation of misaligned chromosomes (H) and failed segregation (I) in HeLa cells transiently depleted of RITA and control cells. The results are from four independent experiments (n = 4, 100 mitotic cells for each condition in each experiment) and presented as mean ± SEM. Student’s t-test was used. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The primers and probes for GAPDH (Hs02758991_g1) and RITA (Hs03044851_m1) were obtained from Thermo Fisher Scientific (Dreieich).

Techniques: Knockdown, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Marker, Microscopy, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Involvement of RBP-J interacting and tubulin-associated protein in the distribution of protein regulator of cytokinesis 1 in mitotic spindles

doi: 10.3389/fcell.2024.1472340

Figure Lengend Snippet: FLAG-RITA precipitates PRC1 and tubulin. (A) Mass spectrometry re-analysis of HA-tagged RITA immunoprecipitates from HEK293T cells revealed potential interaction partners of RITA. Potential partners with a weighted D score (WD score) ≥ 1 are presented. RBP-J, recombination signal binding protein for immunoglobulin kappa J region; PRC1, protein regulator of cytokinesis 1; NuMA, nuclear mitotic apparatus; MAP1B, microtubule-associated protein 1B; LPP, lipoma-preferred partner. (B) HEK293T cells were transiently transfected with FLAG-empty vector or FLAG-RITA plasmids for 48 h and cellular lysates were prepared for immunoprecipitation with tubulin antibody. Precipitates were analyzed by Western blot analysis with indicated antibodies. Left panel: input control. GAPDH served as loading control. Right panel: precipitates were analyzed via Western blot using antibodies as indicated. Antibody light chain was used as reference for precipitate loading. (C) HEK293T cells, transiently transfected as in (B) for 48 h, were treated with DMSO, 10 µM of nocodazole or 10 µM of paclitaxel for 30 min. Cellular lysates were prepared for immunoprecipitation with tubulin antibody. Precipitates were analyzed by Western blot analysis with indicated antibodies. Left panel: input control. β-actin served as loading control. Right panel: precipitates were analyzed via Western blot using indicated antibodies. Antibody heavy chain was used as reference for precipitate loading. (D) HEK293T cells were treated as in (C) and cellular lysates were prepared for immunoprecipitation with anti-FLAG antibody. Precipitates were analyzed by Western blot analysis with indicated antibodies. Left panel: input control. GAPDH served as loading control. Right panel: precipitates were analyzed via Western blot using indicated antibodies. Antibody heavy chain was used as reference for precipitate loading. (E) Subcellular localization of GFP-RITA and PRC1. HeLa CRISPRi sgRITA cells were transiently transfected with GFP-RITA for 48 h and cells were stained for PRC1 (red) and DNA (DAPI, blue) for microscopy. Representative confocal images are shown. Scale: 10 µm. Insets: magnified regions. Inset scale: 10 µm. (F) GFP-RITA transfected HeLa CRISPRi cells were stained for PRC1 (red) and DNA (DAPI, blue) and the co-localization of both proteins in mitotic spindles, the central spindle and the midbody was evaluated via Pearson correlation coefficient, where +1 equates to total positive linear correlation. The Pearson correlation coefficient is indicated as numbers at the column bottom. The correlation assay was independently performed three times (n = 3, 10 for each condition in each experiment) and the results are presented as bar scatter blots, mean ± SD. Student’s t-test was used. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The primers and probes for GAPDH (Hs02758991_g1) and RITA (Hs03044851_m1) were obtained from Thermo Fisher Scientific (Dreieich).

Techniques: Mass Spectrometry, Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Staining, Microscopy, Two-Photon Excitation Fluorescence Cross-Correlation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Involvement of RBP-J interacting and tubulin-associated protein in the distribution of protein regulator of cytokinesis 1 in mitotic spindles

doi: 10.3389/fcell.2024.1472340

Figure Lengend Snippet: Reduced RITA enlarges the PRC1 staining area in mitotic spindles and is associated with decreased inter-centromere distance. (A) HeLa CRISPRi sgRITA and their control cells were stained for α-tubulin (green), PRC1 (red) and DNA (DAPI, blue) for microscopy. Representative confocal images of prometaphase and metaphase cells are shown. Scale: 7.5 µm. (B, C) The staining area of PRC1 was evaluated in the mitotic spindle in prometaphase (B) and metaphase (C) (n = 3, 30 cells for each condition in each experiment). The results are presented as scatter plots with mean ± SD. Mann-Whitney U test was performed. *** p < 0.001. (D, E) HeLa CRISPRi sgRITA and control cells were stained for ACA, α-tubulin and DNA. The inter-centromere distance was measured using the LAS AF software (n = 3, 94–149 pairs for each condition and each experiment). Representative cells are shown (D) . Scale bar: 7.5 μm. Insets: paired centromeres. The results are presented as bar scatter plots, mean ± SD (E) . Mann-Whitney U test was performed. ***p < 0.001. (F–H) HeLa cells, transiently depleted of RITA with siRNA#1 or #2 for 48 h, were stained as indicated in (D) . (F) The inter-centromere distance was evaluated (n = 3, 188–197 pairs for each condition and each experiment) and the results are presented as bar scatter plots, mean ± SD. Mann-Whitney U test was performed. ***p < 0.001. (G) Cellular lysates from treated HeLa cells were prepared for Western blot analysis with RITA antibody. GAPDH served as a loading control. (H) The average protein levels of RITA normalized to the loading control in HeLa cells treated with siRNAs. The results are from three independent experiments.

Article Snippet: The primers and probes for GAPDH (Hs02758991_g1) and RITA (Hs03044851_m1) were obtained from Thermo Fisher Scientific (Dreieich).

Techniques: Staining, Control, Microscopy, MANN-WHITNEY, Software, Western Blot