cag-yap-5sa construct  (Addgene inc)

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Western blot demonstrating the impact of FAT1 knockdown on the Src–Mek–Erk signalling axis in hTERT RPE-1 and T2P cells. The results are representative of three repeats. b , Scatter plot showing the impact of transient siRNA depletion of FAT1 on nuclear YAP1 localization using the stringent PTEMF fixation buffer in TERT RPE-1 cells. The red bars represent median values. Statistical significance was determined by two-sided Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Over 170 cells were scored per condition over three biological replicates. c , Top, schematic illustrating the predicted domains of the FAT1 protein and respective regions cloned into a pCMV expression plasmid with an HA epitope tag. Bottom, TEAD activity in FAT1 knockout PC9 hexaploid WGD cells. The normalized TEAD activity was measured as an enrichment of the neonGreen signal over the untransfected background signal in each experiment. The HA signal was used to identify successful FAT1 rescue construct co-transfection at the single-cell level. TEAD activity was elevated in FAT1 -knockout cells but could be further increased by overexpressing the constitutively active HA–YAP1 5SA mutant. Overexpression of the FAT1 wild-type construct repressed TEAD activity. However, overexpression of FAT1 mutants devoid of the MIB2 binding region (mScarlet–HA–FAT1 MIB∆ ) and HA–FAT1 ICD did not repress TEAD activity. The edges of the histograms represent mean values. Statistical significance was determined by two-sided Kruskal–Wallis test followed by Dunn’s multiple comparisons test. More than 95 cells were scored over three biological replicates. d , DSB resection assay, showing that transient knockdown of LATS1 and LATS2 —both negative regulators of YAP1—represses ssDNA formation at DSB break sites (chr22:37194035, ssDNA measured 131 nucleotides from the DSB) in U2OS-AsiSI cells. The data represent means ± s.d. ( n = 4 biological replicates). Statistical significance was determined by two-sided paired t -test. e , Both LATS1 and LATS2 siRNA knockdown in U2OS cells elevate rates of 53BP1 nuclear body formation when challenged with replication stress (5 h of 4 mM hydroxyurea followed by 24 h recovery). The boxes represent interquartile ranges, the black and red lines represent median and mean values, respectively, and the ranges of the whiskers denote 1.5× the interquartile range. Statistical significance was determined by two-sided Wilcoxon rank-sum test. More than 340 cells were scored over three biological replicates. f , Both LATS1 and LATS2 siRNA knockdown in U2OS cells induce centromeric and acentric micronuclei formation following challenge with replication stress (5 h of 4 mM hydroxyurea followed by 24 h recovery), suggestive of a mitotic segregation deficiency. Statistical significance was determined by repeated measures one-way ANOVA. The data represent means ± s.d. ( n = 3 biological replicates). g , Transient siRNA knockdown of LATS1 in U2OS cells elevates the mitotic error rate. The data represent means ± s.d. Statistical significance was determined by repeated measures one-way ANOVA ( n = 3 biological replicates). MIB2, MindBomb2-interacting domain; mS, mScarlet; TM, transmembrane domain.

Article Snippet: The YAP 5SA construct was obtained from Addgene (plasmid 27371) and subcloned into a pCMV-SPORT6 expression plasmid containing a His-HA tag at the N terminus of the open reading frame.

Techniques: Western Blot, Knockdown, Clone Assay, Expressing, Plasmid Preparation, Activity Assay, Knock-Out, Construct, Cotransfection, Mutagenesis, Over Expression, Binding Assay, Resection Assay

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Impact of FAT1/YAP1 siRNA co-depletion in U2OS cells, as determined by DR-GFP HR reporter assay. MRE11A siRNA served as a positive control. The HR efficiencies are normalized to those of the control samples. Statistical significance was determined by one-way ANOVA with Holm–Šidák correction. The data represent means ± s.d. Biological replicates: n = 5 (siCTRL and siFAT1), n = 3 (siYAP1) and n = 4 (siFAT1 + siYAP1 and siMRE11A). b , ssDNA resection rates for FAT1 / YAP1 siRNA co-depletion, as determined by DIvA U2OS-AsiSI site-directed resection assay with the DSB site located at chr22:37194035, ssDNA measured 131 nucleotides from the DSB. Statistical significance was determined by one-way ANOVA with Holm–Šidák correction. The data represent means ± s.d. ( n = 3 biological replicates). c , Box plots quantifying RAD51 foci formation in A549 cells following the loss of FAT1 , or the combined loss of both FAT1 and YAP1 , after 6 Gy ionizing irradiation and 1 h recovery. The boxes represent interquartile ranges, the black and red lines represents median and mean values, respectively, and the ranges of the whiskers denote 1.5× the interquartile range. Over 380 cells were scored across three biological replicates. Statistical significance was determined by uncorrected Dunn’s test. d , e , Plots ( d ) and representative images ( e ) illustrating the quantification of mitotic error rates in A549 cells after 24 h of aphidicolin treatment (0.2 µM). FAT1 wild-type or knockout cells were transiently depleted of YAP1 using RNAi. For the mitotic error analysis, statistical significance was determined by one-way ANOVA with Holm–Šidák multiple correction and the data represent means ± s.d. (biological repeats: n = 3 ( FAT1 WT) and n = 4 ( FAT1 knockout)). For the DAPI bridge and Fanconi anaemia complementation group D2 (FANCD2)-flanked DAPI bridge analyses, the red lines represent mean values, the boxes represent interquartile ranges and the ranges of the whiskers denote 1.5× the interquartile range, and statistical significance was determined by Dunn’s test with Bonferroni correction. Over 100 mitotic cells were scored across three biological replicates. Scale bars, 5 µM. f , Results of live-cell imaging analysis, showing that FAT1 / YAP1 double siRNA knockdown in TERT–RPE-1 cells fully rescued the failed cytokinesis and WGD introduced by FAT1 knockdown (left) but only partially ameliorated the nuclear shape deformation (right). Statistical significance was determined by one-way ANOVA. At least five biological replicates were quantified per condition. Biological repeats: n = 4 (siCTRL), n = 7 (siFAT1) and n = 5 (siFAT1 + siYAP1). The data represent means ± s.e.m. g , Histogram illustrating the WGD populations in TP53 wild-type versus knockout RPE-1 cells with or without transient mScarlet–YAP 5SA transfection. The data represent means ± s.e.m. Statistical significance was determined by two-sided Mann–Whitney test ( n = 4 biological repeats). h , Histograms illustrating the total (left) and normalized (right) EdU + WGD populations in FAT1 wild-type versus knockout PC9 cells, with or without transient mScarlet–YAP 5SA transfection. The data represent means ± s.e.m. ( n = 5 biological repeats). Statistical significance was determined by repeated measures one-way ANOVA with Benjamini–Hochberg correction (left) or Friedman test (right). KO, knockout.

Article Snippet: The YAP 5SA construct was obtained from Addgene (plasmid 27371) and subcloned into a pCMV-SPORT6 expression plasmid containing a His-HA tag at the N terminus of the open reading frame.

Techniques: Reporter Assay, Positive Control, Control, Resection Assay, Irradiation, Knock-Out, Live Cell Imaging, Knockdown, Transfection, MANN-WHITNEY