yes1 sh yes1  (Addgene inc)

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: YES1 Is a Druggable Oncogenic Target in SCLC.

doi: 10.1016/j.jtho.2022.08.002

Figure Lengend Snippet: Figure 2. Correlation between CNV/mRNA expression and biologic effects elicited by YES1 depletion in vitro. (A) Positive correlation between YES1 mRNA expression of YES1 and CNV in a panel of 45 SCLC cell lines from the CCLE. Relative CNV corresponds to log2 CNV/2, the red line defines CNV gains/amplifications (CNV 2.5) and the blue line defines CNV deletions (CNV 0.8). (B) IHC and FISH images illustrating YES1 expression and CNV in DMS53 and H209 tumor xenografts. Scale bars: 50

Article Snippet: Silencing of YES1 was achieved with short hairpin RNAs (shRNAs) (Sigma-Aldrich), whose sequences can be found in Supplementary Table 2. shRNAs targeting YES1 (sh-YES1) were cloned into the lentiviral TetpLKO-puro plasmid (Addgene #21915). shRNA against GFP (shGFP) was used as control and lentiviral particles for both shRNAs were produced as previously described.27 Cell lines were infected by adding lentiviral particles containing the plasmid and polybrene (8 mg/ mL) and centrifuging them at 1200 revolutions per minute for 2 hours at 32oC.

Techniques: Expressing, In Vitro

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: YES1 Is a Druggable Oncogenic Target in SCLC.

doi: 10.1016/j.jtho.2022.08.002

Figure Lengend Snippet: Figure 3. Inhibition of YES1 abrogates tumor growth and metastasis. (A) Tumor growth of DMS53 shGFP (n ¼ 7) and sh-YES1 (n ¼ 7 per shRNA) cells subcutaneously implanted in immunosuppressed mice and treated with doxy when the tumors reached an initial volume of 100 mm3 to induce the shRNA expression. (B) Waterfall plot illustrating the tumor volume change of DMS53 control and YES1-inhibited tumors at day 33. (C) Mice OS curve corresponding to the experiment illustrated in A and B.

Article Snippet: Silencing of YES1 was achieved with short hairpin RNAs (shRNAs) (Sigma-Aldrich), whose sequences can be found in Supplementary Table 2. shRNAs targeting YES1 (sh-YES1) were cloned into the lentiviral TetpLKO-puro plasmid (Addgene #21915). shRNA against GFP (shGFP) was used as control and lentiviral particles for both shRNAs were produced as previously described.27 Cell lines were infected by adding lentiviral particles containing the plasmid and polybrene (8 mg/ mL) and centrifuging them at 1200 revolutions per minute for 2 hours at 32oC.

Techniques: Inhibition, shRNA, Expressing, Control

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: YES1 Is a Druggable Oncogenic Target in SCLC.

doi: 10.1016/j.jtho.2022.08.002

Figure Lengend Snippet: Figure 4. Pharmacologic inhibition of YES1 impairs cell proliferation and targets phospho-FAK tyrosine kinase. (A, B) Cell proliferation curves illustrating the IC50 for dasatinib (A) and CH6953755 (B) in DMS53, H209, and H69 cell lines. (C) H&E staining of DMS53-, H209- and PDX YU-16-derived organoids. Scale bar: 5 mm. (D–F) Growth curves of DMS53- (D), H209- (E), and YU-16-derived organoids (F) in the presence of dasatinib or CH6953755. (G) Representative images of untreated PDX YU-

Article Snippet: Silencing of YES1 was achieved with short hairpin RNAs (shRNAs) (Sigma-Aldrich), whose sequences can be found in Supplementary Table 2. shRNAs targeting YES1 (sh-YES1) were cloned into the lentiviral TetpLKO-puro plasmid (Addgene #21915). shRNA against GFP (shGFP) was used as control and lentiviral particles for both shRNAs were produced as previously described.27 Cell lines were infected by adding lentiviral particles containing the plasmid and polybrene (8 mg/ mL) and centrifuging them at 1200 revolutions per minute for 2 hours at 32oC.

Techniques: Inhibition, Staining, Derivative Assay

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: YES1 Is a Druggable Oncogenic Target in SCLC.

doi: 10.1016/j.jtho.2022.08.002

Figure Lengend Snippet: Figure 6. YES1 protein is released in exosomes and can be detected in SCLC and NSCLC plasma-derived exosomes. (A) Schematic representation of the protocol followed for the exosomes’ extraction from conditioned DMS53 and H209 culture media, plasma from the mouse models or plasma from patients with lung cancer. (B) Western blotting of the exosomal markers Alix, TSG101, CD63, and CD9, and YES1 in DMS53 and H209 cell lysates and their derived exosomes isolated from the conditioned media after 96 h. Electron microscopy images of DMS53 and H209 exosomes. (C) Western blotting illustrating the decrease in YES1 protein levels in exosomes isolated from plasmas of mice carrying shGFP (n ¼ 2), sh1-YES1 (n ¼ 1), or sh2-YES1 (n ¼ 1) H209 subcutaneous tumors. (D) Western blotting of pSFKs, and exosomal markers in plasma-derived exosomes from mice implanted with PDX YU-16 and treated with CH6953755 or dasatinib. (E) Protein expression of exosomal markers and YES1 in NSCLC patient-derived exo- somes and representative images of YES1 IHC in some of those NSCLC specimens. A representative electron microscopy image of exosomes from a patient with NSCLC is also illustrated. (F) Protein levels of the exosomal markers mentioned above and YES1 in six SCLC patient-derived exosomes and a healthy donor. Representative images of YES1 protein levels in SCLC specimens and an electron microscopy imageof exosomes isolated froma patient with SCLC. Scale barin IHC: 50mm.Scale barin exosomes images: 100 nm. h, hour; H, high; IHC, immunohistochemistry; KD, knockdown; L, low; M, medium; pSFK, phospho-SFK; sh1-YES1, shRNA 1 targeting YES1; sh2-YES1, shRNA 2 targeting YES1; shGFP, shRNAs targeting GFP; shRNA, short hairpin RNA.

Article Snippet: Silencing of YES1 was achieved with short hairpin RNAs (shRNAs) (Sigma-Aldrich), whose sequences can be found in Supplementary Table 2. shRNAs targeting YES1 (sh-YES1) were cloned into the lentiviral TetpLKO-puro plasmid (Addgene #21915). shRNA against GFP (shGFP) was used as control and lentiviral particles for both shRNAs were produced as previously described.27 Cell lines were infected by adding lentiviral particles containing the plasmid and polybrene (8 mg/ mL) and centrifuging them at 1200 revolutions per minute for 2 hours at 32oC.

Techniques: Clinical Proteomics, Derivative Assay, Extraction, Western Blot, Isolation, Electron Microscopy, Expressing, Immunohistochemistry, Knockdown, shRNA