Journal: Advanced Science

Article Title: A Novel tsRNA, m 7 G‐3′ tiRNA Lys TTT , Promotes Bladder Cancer Malignancy Via Regulating ANXA2 Phosphorylation

doi: 10.1002/advs.202400115

Figure Lengend Snippet: mtiRL enhances the binding between Yes1 and ANXA2. A,B) Endogenous Co‐immunoprecipitation (co‐IP) assays revealed that Yes1 bound to ANXA2. C,D) Exogenous co‐IP assays confirmed that GFP‐Yes1 interacted with Flag‐ ANXA2. E) Colocalization of Yes1 and ANXA2 was confirmed by immunofluorescence staining in Cd‐SV‐HUC‐1 cells. F–I) WB results showed that overexpression of Yes1 upregulated the level of p‐ANXA2‐Y24, whereas it did not affect the expression of total ANXA2 protein. J–M) Co‐IP experiments in Cd‐SV‐HUC‐1 cells revealed that the interaction between endogenous Yes1 and ANXA2 was significantly diminished by knockdown of mtiRL. N) In vitro kinase assay indicated that active GST‐Yes1 could phosphorylate ANXA2 at Tyr24 site and mtiRL enhance the phosphorylation of Yes1. * p < 0.05, ** p < 0.01.

Article Snippet: [ ] Recombinant human His‐ANXA2 (100 ng; Jingxin Bio, #GXP87073) was incubated with recombinant human active GST‐Yes1 (100 ng; MCE, # HY‐P73486) and mtiRL (1 μg) at 30 °C for 30 min in reaction buffer (20 mM HEPES (pH 7.6), 20 mM MgCl 2 , 0.2 mM ATP, 2 mM DTT, 20 mM β‐glycerophosphate, 0.1 mM sodium orthovanadate).

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Over Expression, Expressing, Knockdown, In Vitro, Kinase Assay, Phospho-proteomics

Journal: Frontiers in Genetics

Article Title: Destabilized 3’UTR elements therapeutically degrade ERBB2 mRNA in drug-resistant ERBB2+ cancer models

doi: 10.3389/fgene.2023.1184600

Figure Lengend Snippet: Engineered destabilized 3’UTR of ERBB2 degrades specific kinases implicated in ERBB2+ trastuzumab-resistant breast cancer and in ERBB2-expressing osimertinib-resistant EGFR790M non-small-cell lung cancer cells. (A) Dot blots show the phospho-kinase array pattern of the BT474 clone 5 wildtype, BT474 clone 5 vector, BT474 clone 5 desARE3’UTR ERBB2-3, and BT474 clone 5 desARE3’UTR c-MYC 2–3. The dot marked in the red box shows unique kinases downregulated only in BT474 clone 5 desARE3’UTR ERBB2-3 and the dot marked in green boxes shows unique kinases downregulated only in BT474 clone 5 desARE3’UTR c-MYC 2-3. Each kinase is spotted twice. (B) Bar chart shows the intensity of the phospho-kinase array dot blot for the BT474 clone 5 wildtype (black bar), BT474 clone 5 vector (blue bar), BT474 clone 5 desARE3’UTR ERBB2-3 (red bar), and BT474 clone 5 desARE3’UTR c-MYC 2-3 (green bar). (C) Bar charts show YES1 pT60 and WNK1 pT60 kinases specifically downregulated in BT474 clone 5 desARE3’UTR ERBB2-3 in red bar. Controls BT474 clone 5 wildtype (black bar), BT474 clone 5 vector (blue bar), and BT474 clone 5 desARE3’UTR c-MYC 2-3 (green bar). T-test (*** p -value = 0.00413, WT, vector vs. desARE3’UTR ERBB2-3 for YES and WNK1). (D) Venn diagram shows kinases downregulated in destabilized desARE3’UTR ERBB2-3 and -30 NCI- H1975 (p = 0.02) blue (from RNA Seq), and in destabilized desARE3’UTR ERBB2-3 BT474 clone 5 (red-kinase array) and the shared kinases found downregulated in destabilized ERBB2 in both resistant cell lines WNK1 and YES1 in pink.

Article Snippet: The YES1 Y537F overexpression vector (Addgene: 51299) was transfected into desARE3’UTR ERBB2-3 and -30 cells that have lost ERBB2 and YES1 by introduction of destabilized desARE3’UTR ERBB2.

Techniques: Expressing, Plasmid Preparation, Dot Blot, RNA Sequencing

Journal: Frontiers in Genetics

Article Title: Destabilized 3’UTR elements therapeutically degrade ERBB2 mRNA in drug-resistant ERBB2+ cancer models

doi: 10.3389/fgene.2023.1184600

Figure Lengend Snippet: Validation of the engineered destabilized 3’UTR of ERBB2 degradation of YES1 and WNK1 and inhibition of cancer cell migration and induction of caspase 7 in vivo in treated tumor-bearing mice. (A) Western blot show YES1 and GAPDH expression across the NCI-H1975 wildtype cells, vector, cells containing constructs desARE3’UTR ERBB2-1, -3, and -30, and wildtype trastuzumab-treated NCI-H1975 cells (n = 2). (B) Bar chart shows quantification of the YES1 expression normalized against GAPDH on NCI-H1975 wildtype cells, vector, and cells containing constructs desARE3’UTR ERBB2-1, -3, and -30 (n = 2). T-test (*** p -value = 0.0021, WT, vector vs. desARE3’UTR ERBB2-1, -3, and -30). (C) Western blot shows WNK1 and GAPDH expression across the NCI-H1975 wildtype cells, vector, cells containing constructs desARE3’UTR ERBB2-1, -3, and -30 and wildtype trastuzumab-treated NCI-H1975 cells. (D) Western blot shows YES1 and GAPDH expression across the NCI-H1975 destabilized with constructs desARE3’UTR ERBB2-3 and -30 (lanes 1 and 3) and over expressed with YES1 in lanes 2 and 4. (E) Bar charts show the wound healing closing of NCI-H1975 wildtype, vector, desARE3’UTR ERBB2-1, -3, -30, and desARE3’UTR ERBB2-30,30 overexpressed with YES1 from 0 h to 72 hrs. At 24hrs ***p = 0.00025 WT, vector vs. desARE3’UTR ERBB2-3,30. At 48 hrs ****p = 0.000018 WT, vector vs. desARE3’UTR ERBB2-3 and at 72 hrs ***p = 0.0003 WT, vector vs. desARE3’UTR ERBB2-3 and **p = 0.002 WT, vector vs. desARE3’UTR ERBB2-30. (F) Pictures depict caspase 7 expression in vivo in tumors untreated NCI-H1975 wildtype, vector, desARE3’UTR ERBB2-1, -3, and -30 and quantified in bar charts. Magnification is 60x.

Article Snippet: The YES1 Y537F overexpression vector (Addgene: 51299) was transfected into desARE3’UTR ERBB2-3 and -30 cells that have lost ERBB2 and YES1 by introduction of destabilized desARE3’UTR ERBB2.

Techniques: Biomarker Discovery, Inhibition, Migration, In Vivo, Western Blot, Expressing, Plasmid Preparation, Construct

Journal: Frontiers in Genetics

Article Title: Destabilized 3’UTR elements therapeutically degrade ERBB2 mRNA in drug-resistant ERBB2+ cancer models

doi: 10.3389/fgene.2023.1184600

Figure Lengend Snippet: Comparison of ShRNAi, CRISPR, and mRNA 3’UTR destabilization in targeting HER2 in colorectal carcinoma HCT116.

Article Snippet: The YES1 Y537F overexpression vector (Addgene: 51299) was transfected into desARE3’UTR ERBB2-3 and -30 cells that have lost ERBB2 and YES1 by introduction of destabilized desARE3’UTR ERBB2.

Techniques: Comparison, CRISPR, Genome Wide, shRNA

Journal: Science Advances

Article Title: FeaSion decodes the regulatory landscape and functional diversity of RNA polymerase II CTD phosphorylation

doi: 10.1126/sciadv.adz2345

Figure Lengend Snippet: ( A ) Stacked bar plot showing the kinases (left) and phosphatases (right) identified in RNAPII ChIP-MS, CRISPR screening and RNAPII mut ChIP-MS. ( B ) Pie plots showing the family of kinases identified in CRISPR screening. ( C ) Heatmap showing the NormZ of CRISPR screening for CLK1, CLK4, and YES1. ( D ) Western blot analyses of the RNAPII, pY1, pS2, pT4, pS5, and pS7 in CLK1, CLK4, or YES1 KO cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. ( E ) Metagene profiles and boxplots (top) and log 2 (fold change) metagene heatmaps (bottom) showing pS7 and RNAPII ChIP-seq density in control, CLK1 KO, and CLK4 KO cells, as well as in untreated and TG003-treated (20 μM, 4 hours) cells (top). Statistical significance was assessed by a two-sided Wilcoxon test. *** P < 0.001. The value on the box plot indicating the fold change. ( F ) Similar to (E), but for pY1 and RNAPII ChIP-seq in control and YES1 KO cells, as well as in untreated and CHR6953755-treated (10 μM, 24 hours) cells. ( G ) Schematic workflow of in vitro phosphorylation assay. ( H ) In vitro phosphorylation activity of CLK1 (left), CLK4 (middle), and YES1 (right) on CTD. The CTD was detected using HA tag, and CLK1, CLK4, and YES1 were detected using Flag tag. ( I ) Pie chart (top) and metagene heatmap (bottom) showing the distribution of the top 500 CLK1 (left) CLK4 (middle) and YES1 (right) ChIP-seq binding sites and their densities. ( J ) Bar plot showing the gene ontology enrichment result of CLK1 (top), CLK4 (middle) and YES1 (bottom) binding genes. ( K ) Tracks of ING5 of CLK1 (left), DLX2 of CLK4 (middle), and IMPACT of YES1 (right) ChIP-seq signals and the changes of pS7 or pY1 and RNAPII after their knockout. miRNA, microRNA; snoRNA, small nucleolar RNA. TES, transcription end site.

Article Snippet: The supernatant was collected and incubated with the RNPII antibody (Santa Cruz Biotechnology, #sc-55492), phospho-RPB1 CTD (Ser 2 ) antibody (Cell Signaling Technology, #13499), phospho-RPB1 CTD (Ser 5 ) antibody (Cell Signaling Technology, #13523), phospho-RPB1 CTD (Tyr 1 ) antibody (Merck Millipore #MABE350), phospho-RPB1 CTD (Thr 4 ) antibody (Cell Signaling Technology, #14934), phospho-RPB1 CTD (Ser 7 ) antibody (Cell Signaling Technology, #13780), H3K36me3 antibody (Abcam, #ab9050), acetyl-histone H2A.Z (Lys 4 /Lys 7 ) antibody (Cell Signaling Technology, #75336), CLK1 antibody (Santa Cruz Biotechnology, #sc-515897), CLK4 antibody (Proteintech, #31205-1-AP), or YES1 antibody (Proteintech, #20243-1-AP) overnight at 4°C on a rotator.

Techniques: CRISPR, Western Blot, Control, ChIP-sequencing, In Vitro, Phospho-proteomics, Activity Assay, FLAG-tag, Binding Assay, Knock-Out

Journal: Nature cardiovascular research

Article Title: Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis.

doi: 10.1038/s44161-022-00172-z

Figure Lengend Snippet: Fig. 2 | Yes phosphorylates VE-cadherin. a–c, Immunofluorescence of Yes1 iECKO (Yes1fl/fl, Cdh5CreERT2+) and control (Yes1fl/fl, Cdh5CreERT2−) P6 retinas showing total VE-cadherin (magenta) and phosphorylated VE-cadherin (green) for pY685 (a), pY658 (b) and pY731 (c). Scale bars, 50 µm. d–f, Quantification of pVE-cadherin (pY685) (d), pVE-cadherin (pY658) (e) and pVE-cadherin (pY731) (f) levels normalized to total VE-cadherin in control and Yes1 iECKO littermate P6 retinas. Control, n = 5; Yes1 iECKO, n = 4. g–i, Quantification of pVE-cadherin levels normalized to total VE-cadherin in P6 retinas of Src iECKO (Srcfl/fl, Cdh5CreERT2+) and control (Srcfl/fl, Cdh5CreERT2−) mice. pVE-cadherin (pY685) (g). Control, n = 8; Src iECKO, n = 10. pVE-cadherin (pY658) (h). Control, n = 5; Src iECKO, n = 5. pVE-cadherin (pY731) (i). Control, n = 7; Src iECKO, n = 10. j, Immunofluorescence showing the correlation between levels of pY685 VE- cadherin and YFP+ Yes1-deficient ECs in a P6 retina with chimeric recombination (100 µg tamoxifen per mouse) at P3. Arrows, YFP− ECs; arrowheads, YFP+ ECs.

Article Snippet: Conditional Yes1 knock-in mouse H11-CAG-STOP-Yes1 was generated using CRISPR/ CAS (Taconic/Cyagen; Extended Data Fig. 4a).

Techniques: Immunofluorescence, Control

Journal: Nature cardiovascular research

Article Title: Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis.

doi: 10.1038/s44161-022-00172-z

Figure Lengend Snippet: Fig. 3 | Disturbed collective EC migration in Yes deficiency. a, Timeline for EC distribution analysis (100 µg tamoxifen per mouse at P3). b, Schematics showing two-dimensional maps of fluorescently labeled ECs in flat-mounted retinas. x axis; relative distance between veins (0.0) and arteries (1.0); y axis; distance (µm) from optic nerve to sprouting front. c, Distribution analysis of ECs with Cdh5Cre- induced expression of MbTomato (green) in Yes1wt/wt, iSuRe-Cre+, Cdh5CreERT2+ and Yes1fl/fl, iSuRe-Cre+, Cdh5CreERT2+ at P7 (left) and P15 (right). CD31 (magenta) counterstaining shows all ECs. Boxed regions with veins and arteries shown enlarged (center). Distribution analysis (right) showing control (orange) and Yes1 iECKO (blue) ECs. Color density indicates average density of MbTomato+ cells in six retinas. Scale bars, 500 µm. d, Histogram plots of vein-to-artery distribution of MbTomato+ ECs in P7 and P15 retinas. Solid lines indicate median position of total MbTomato+ ECs detected. Dashed lines indicate positions of 25% and 75% of total MbTomato+ ECs. e, Box plot showing mean values of relative vein-to-artery distance of all MbTomato+ ECs/retina. Minima, maxima and center bounds show

Article Snippet: Conditional Yes1 knock-in mouse H11-CAG-STOP-Yes1 was generated using CRISPR/ CAS (Taconic/Cyagen; Extended Data Fig. 4a).

Techniques: Migration, Labeling, Expressing, Control

Journal: Nature cardiovascular research

Article Title: Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis.

doi: 10.1038/s44161-022-00172-z

Figure Lengend Snippet: Fig. 4 | Adherens junction morphology. a, Schematic outline of linear and jagged adherens junction morphology based on VE-cadherin immunostaining. b, Immunostaining of VE-cadherin in HUVECs transfected with control or YES1 or SRC siRNA and cultured under static or 3 dyn cm−2 flow conditions. Boxed regions show representatives of altered VE-cadherin morphologies, magnified to the lower right. Scale bars, 20 µm. c, Quantification of jagged junctions as shown in b, given as junction length in percentage of total. Bar graphs show mean ± s.e.m. with individual data points. siControl; n = 4, siYES1; n = 4, siSRC;

Article Snippet: Conditional Yes1 knock-in mouse H11-CAG-STOP-Yes1 was generated using CRISPR/ CAS (Taconic/Cyagen; Extended Data Fig. 4a).

Techniques: Immunostaining, Transfection, Control, Cell Culture

Journal: Nature cardiovascular research

Article Title: Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis.

doi: 10.1038/s44161-022-00172-z

Figure Lengend Snippet: Fig. 6 | Cell–cell junctions and actin organization. a,b, Endothelial junctions in control, Yes1 iECKO or Src iECKO mouse ear dermis vessels after intradermal injection of saline or VEGFA164, visualized by TEM. EC junction length is highlighted in green and electron-dense junctional area (cortical actin-rich domain) is in red. Scale bars, 500 nm. c, Quantification of actin area/junction length in control (Yes1fl/fl, Cdh5CreERT2−) and Yes1 iECKO mice. Saline-treated control mice, n = 9; VEGFA-treated control mice, n = 7; saline-treated Yes1 iECKO mice, n = 10; VEGFA-treated Yes1 iECKO mice, n = 11. Overall, 2–40 junctions

Article Snippet: Conditional Yes1 knock-in mouse H11-CAG-STOP-Yes1 was generated using CRISPR/ CAS (Taconic/Cyagen; Extended Data Fig. 4a).

Techniques: Control, Injection, Saline