Journal: iScience

Article Title: Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding

doi: 10.1016/j.isci.2025.113743

Figure Lengend Snippet: Construction of FRET calibration standards (A) Construction of FRET-ON calibration standard by Y349F mutation of Cyto-FAK. (B) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to 100 ng/mL EGF added at 6 min. (C) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to FAK inhibitor VS-6063 over 24 h. (D) Construction of FRET-OFF calibration standard by removal of the H3 domain of the H3K9me9 (W45A) biosensor. (E) Plot of YFP/CFP ratio of FRET-OFF and H3K9me3 biosensor (mean ± SEM, n = 20 cells) in response to 5 μM TCP over 48 h.

Article Snippet: H3K9me3 (W45A) biosensor , Addgene , Cat#120808.

Techniques: Mutagenesis

Journal: Science Advances

Article Title: Mechanochemical waves in focal adhesions during cell migration

doi: 10.1126/sciadv.adw6425

Figure Lengend Snippet: ( A ) The FAK biosensor is composed of ECFP, SH2 domain, flexible linker, FAK substrate peptide, YPet, and FAT domain. ( B ) Schematics illustrating the FRET effect of the FAK biosensor upon the actions of FAK phosphorylation or dephosphorylation. Upon phosphorylation of Y397 in the biosensor FAK substrate peptide, the SH2 domain forms an intramolecular complex with the phosphotyrosine side chain, increasing the distance between the FRET pair to alter the FRET signal. Dephosphorylation reverses the FRET change. ATP, adenosine 5′-triphosphate. ( C ) C-terminal FAT domain recruits the biosensor to FAs. YPet intensity showing slight changes before and after FAK inhibition (FAKi; 10 μM PF-573228, 60 min). ( D ) ECFP/FRET signal before and after FAKi (10 μM PF-573228, 60 min) showing that the biosensor is specific and sensitive to FAK activity. ( E ) EFCP/FRET signal at individual FAs [ n = 195 FAs from seven cells across three independent experiments for FAKi (10 μM PF-573228, >60 min); n = 151 FAs from six cells across three independent experiments for DMSO control; means ± SD]. ( F ) Fluorescence lifetime image and quantification for fibroblasts expressing the FAK biosensor ( n = 23 FAs from six cells across three independent experiments). Scale bar, 20 μm. ( G ) FRET efficiency image and quantification for FAs and cytosol ( n = 19 FAs from five cells across three independent experiments). Scale bar, 20 μm. a.u., arbitrary units.

Article Snippet: The FAK FRET biosensor construct is available from Addgene (plasmid no. 78303).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Inhibition, Activity Assay, Control, Fluorescence, Expressing

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: iScience

Article Title: Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding

doi: 10.1016/j.isci.2025.113743

Figure Lengend Snippet: Construction of FRET calibration standards (A) Construction of FRET-ON calibration standard by Y349F mutation of Cyto-FAK. (B) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to 100 ng/mL EGF added at 6 min. (C) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to FAK inhibitor VS-6063 over 24 h. (D) Construction of FRET-OFF calibration standard by removal of the H3 domain of the H3K9me9 (W45A) biosensor. (E) Plot of YFP/CFP ratio of FRET-OFF and H3K9me3 biosensor (mean ± SEM, n = 20 cells) in response to 5 μM TCP over 48 h.

Article Snippet: H3K9me3 biosensor , Addgene , Cat#120802.

Techniques: Mutagenesis