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

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Cfos and cjun mutations detected in synovial tissue of rheumatoid arthritis (RA) and osteoarthritis (OA) patients.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques:

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Effects of cjun and cfos mutations on reporter gene expression. The graph shows pentameric AP-1 site- and matrix metalloproteinase 1 (MMP-1) promoter-dependent expression of firefly luciferase 2 days following transfection of NIH-3T3 cells with wt ( A ) or mutated ( B , C ) cfos and cjun expression plasmids (determined in biological triplicates; means ± standard error of the mean). Firefly luciferase expression levels were normalized to renilla luciferase expression levels in the respective samples (transfection and normalization control). Results are presented as relative luciferase activity (in x-fold) related to the expression level in the presence of the control vector ( A ), cfos wt expression vector ( B ), or cfos and cjun wt vectors ( C ). For normalization and to correct for different transfection efficiencies, the renilla luciferase-expressing vector pRL-CMV was co-transfected. In all analyzed samples, luciferase expression was easy to detect and clearly exceeded background levels in non-transfected cells. * p ≤ 0.05 vs. controls, i.e., pCMX ( A ), fosWt ( B ), and fosWt/junWt ( C ); +, °, §, ‡, and # p ≤ 0.05 vs. respective other mutants; fos73 represents the double mutant fos73/fos125.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques: Gene Expression, Expressing, Luciferase, Transfection, Control, Activity Assay, Plasmid Preparation, Mutagenesis

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: DNA binding activity of AP-1 complexes in transfected K4IM fibroblasts and protein expression of cfos and cjun variants. ( A ) Electrophoretic mobility shift assay (EMSA) of nuclear extracts from transfected K4IM cells. The phosphor-image exemplarily shows binding of radio-labelled AP-1 sites by AP-1 complexes formed in K4IM cells transfected with the indicated cfos and/or cjun variants ( n = 1). ( B ) In the corresponding whole cell extracts, protein amounts of cFos and cJun following transfection were detected by Western Blot ( n = 1). Loading control: glyceraldehyde 3-phosphate dehydrogenase (GAPDH); fos73: double mutant fos73/fos125.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques: Binding Assay, Activity Assay, Transfection, Expressing, Electrophoretic Mobility Shift Assay, Western Blot, Control, Mutagenesis

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Quantitation of MMP-1 mRNA expression in jun-/fos-transfected primary human fibroblast-like synoviocytes (FLS). ( A – C ) The graph shows the MMP-1 mRNA expression (detected by qPCR) in primary FLS derived from RA and OA patients (without detectable genetic variations in the jun and fos genes; n = 3 each, mean ± SD) following transfection of cjun and/or cfos expression vectors. ( A ) MMP-1 expression in FLS transfected with the indicated cfos variants. ( B ) MMP-1 expression in FLS transfected with the indicated cjun variants. ( C ) MMP-1 expression in FLS transfected with combinations of cfos and cjun variants. MMP-1 mRNA levels in fosWt- ( A ), junWt- ( B ), or fosWt/junWt-transfected cells ( C ) were set as 1. Statistical analyses: Mann–Whitney U-test, * p ≤ 0.05 vs. the respective controls; fos73: double mutant fos73/fos125.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques: Quantitation Assay, Expressing, Transfection, Derivative Assay, MANN-WHITNEY, Mutagenesis

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Quantitation of IL-6 mRNA expression in jun-/fos-transfected primary human FLS. ( A – C ) The graph shows the IL-6 mRNA expression (detected by qPCR) in primary FLS derived from RA and OA patients (without detectable genetic variations in the jun and fos genes; n = 3 each, mean ± SD) following transfection of cjun and/or cfos expression vectors. ( A ) IL-6 expression in FLS transfected with the indicated cfos variants. ( B ) IL-6 expression in FLS transfected with the indicated cjun variants. ( C ) IL-6 expression in FLS transfected with combinations of cfos and cjun variants. IL-6 mRNA levels in fosWt- ( A ), junWt- ( B ), or fosWt/junWt-transfected cells ( C ) were set as 1. Statistical analyses: Mann–Whitney U-test, * p ≤ 0.05 vs. the respective controls; fos73: double mutant fos73/fos125.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques: Quantitation Assay, Expressing, Transfection, Derivative Assay, MANN-WHITNEY, Mutagenesis

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Frequency analysis of mutations in GnomAD.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques: