Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A) Representative images of G2 phase hTERT-RPE1 cells expressing cellular Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (B) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from G2 phase cells represented in (A) derived from N=3 independent experiments comprising n= 115: siControl, 124: siBora, 117: siCep192, 122: siCenexin, 132: siCep192 + siCenexin and 125: Plk1i cells. (C) Representative images of G2 phase hTERT-RPE1 cells expressing centrosomal Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (D) Box and Whisker plot for FRET measurement-based quantification of centrosomal Plk1 activity calculated from G2 phase cells represented in (C) derived from N=4 independent experiments comprising n= 183: siControl, 183: siBora, 181: siCep192, 183: siCenexin, 185: siCep192 + siCenexin and 192: Plk1i cells. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars = 5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Whisker Assay, Derivative Assay

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A) Representative images of G2 phase hTERT-RPE1 cells expressing cellular Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs. (B) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from G2 phase cells represented in (A) derived from N=3 independent experiments comprising n= 120: siBora + siCep192, 135: siBora + siCenexin, 127: siBora + siCep192 + siCenexin, 124: siAurora and 121: siPlk1 cells. (C) Representative images of G2 phase hTERT-RPE1 cells expressing centrosomal Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (D) Box and Whisker plot for FRET measurement-based quantification of centrosomal Plk1 activity calculated from G2 phase cells represented in (C) derived from N=4 independent experiments comprising n= 181: siBora + siCep192, 187: siBora + siCenexin, 181: siBora + siCep192 + siCenexin, 182: siAurora and 182: siPlk1 cells. (E-G) Western Blot confirming the depletion of indicated proteins after indicated siRNA treatments. Data represent the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars = 5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Whisker Assay, Derivative Assay, Western Blot

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A-C) Flow cytometry-based quantification of cell cycle profile of hTERT-RPE1 (WT), hTERT-RPE1: Usp28 - / - and hTERT-RPE1: Usp28 - / - 0:0 cells revealing percentage of cells in G1 (A) , S (B) and G2 (C) phases derived from N=4 independent experiments with each treatment comprising around 30,000 cells. (D) Flow cytometry-based quantification of cell cycle profile or RPE1 cells treated with Control vs Cep192 siRNA showing percentage of cells in G1, S and G2 phases obtained from N=5 independent experiments with each treatment comprising of 30,000 cells. (E) Representative images of S phase hTERT-RPE1 cells expressing cellular Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (F) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from S phase cells represented in (E) derived from N=4 independent experiments comprising n= 203: siControl, 204: siBora, 215: siCep192, 219: siCenexin, 215: Plk1i cells. The leftmost G2 Phase – siControl bar shown is replica of siControl data shown in for side-by-side comparison purposes. G) Representative images of S phase hTERT-RPE1 cells expressing centrosomal Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (H) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from S phase cells represented in (G) derived from N=3 independent experiments comprising n= 163: siControl, 187: siBora, 187: siCep192, 184: siCenexin, 178: Plk1i cells. The leftmost G2 Phase – siControl bar shown is replica of siControl data shown in for side-by-side comparison purposes. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars, Images: 5 µm and zoomed insets: 0.5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Flow Cytometry, Derivative Assay, Control, Expressing, Activity Assay, Whisker Assay, Comparison

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A) Representative images of G2 phase centrosomes stained with γ-tubulin (red) and Pericentrin (green) in hTERT-RPE1 cells treated with indicated siRNA combinations. (B-C) Box and Whisker plots representing the centrosomal levels of γ-tubulin (B) and Pericentrin (C) in cells represented in (A) as derived from N=3 independent experiments consisting of n= (138: siBora + siCep192, 164: siCep192 + siCenexin, 181: siCenexin + siBora and 167: siBora + siCep192 + siCenexin for γ-tubulin) and (n= 136: siBora + siCep192, 167: siCep192 + siCenexin, 181: siCenexin + siBora and 170: siBora + siCep192 + siCenexin for Pericentrin (green) cells. Data presented as the entire range with values between the first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars: 0.5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Staining, Whisker Assay, Derivative Assay

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A) Representative images of G2 phase centrosomes stained with γ-tubulin (red) and Pericentrin (green) in hTERT-RPE1 cells treated with indicated siRNAs. (B-C) Box and Whisker plots representing the centrosomal levels of γ-tubulin (B) and Pericentrin (C) in cells represented in (A) as derived from N=3 independent experiments consisting of n= (168: siControl, 171: siBora, 177: siCep192, 165: siCenexin and 168: siPlk1 for γ-tubulin) and (n= 176: siControl, 170: siBora, 179: siCep192, 157: siCenexin and 183: siPlk1 for Pericentrin (green) cells. Data presented as the entire range with values between the first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars: 0.5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Staining, Whisker Assay, Derivative Assay

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A) Expansion microscopy images of centrioles in G2 phase hTERT-RPE1 (WT) and hTERT-RPE1 Cenexin - / - cells stained against α-tubulin and treated with indicated siRNAs to visualise centriole configuration. The arrowheads indicate the disengaged mother daughter centriole pairs. (B) Quantification of percentage of G2 phase hTERT-RPE1 cells with disengaged centrioles in their centrosomes ( N = 3 independent experiments, n = 96, 98, 100 and 104 cells in siControl, siBora, siCep192 and siBora + siCep192, respectively) (C) Quantification of percentage of G2 phase hTERT-RPE1 Cenexin - / - cells with disengaged centrioles in their centrosomes ( N = 5 independent experiments, n = 166, 149, 138 and 145 cells in siControl, siBora, siCep192 and siBora + siCep192, respectively) (D) Representative images of hTERT-RPE1: Centrosomal Separase activity sensor cells depicting centrosomal intensity of GFP and mCherry after indicated siRNA/drug treatments. (E) Box and Whisker plot for quantification of centrosomal activity of Separase in conditions depicted in (D) and derived from N=3 independent experiments consisting of n=142: siControl, 144: siBora, 134: siCep192, 152: siCenexin, 152: siSeparase and 147: Aphidicolin treated cells. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars: 0.5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Microscopy, Staining, Activity Assay, Whisker Assay, Derivative Assay