yes1 y537f overexpression vector  (Addgene inc)

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