Additional file 2 of GRK3 is a poor prognosticator and serves as a therapeutic target in advanced gastric adenocarcinoma
Creators: name: Li, Yuan
name: Fan, Yibo
name: Xu, Jinbang
name: Huo, Longfei
name: Scott, Ailing W.
name: Jin, Jiankang
name: Yang, Boxuan
name: Shao, Shan
name: Ma, Lang
name: Wang, Ying
name: Yao, Xiaodan
name: Pool Pizzi, Melissa
name: Sewastjanow Da Silva, Matheus
name: Zhang, Guoliang
name: Zhuo, Lijuan
name: Cho, Eun Jeong
name: Dalby, Kevin N.
name: Shanbhag, Namita D.
name: Wang, Zhenning
name: Li, Wenliang
name: Song, Shumei
name: Ajani, Jaffer A.
Publisher: name: figshare
Year: 2022
Subjects: Biophysics
Biochemistry
Medicine
Cell Biology
Molecular Biology
Pharmacology
Chemical Sciences not elsewhere classified
Biological Sciences not elsewhere classified
Cancer

description: Addiitional file 2: Supplementary Figure1. GRK3 expression in PDX tumors of GAC and paired primary tumor and PC. A. Diagram demonstrates the several patients’ PC cells (malignant ascites cells) into nude mice to generate PDXs (left). Western blot of GRK3 expression of Primary and ascites from PDX tumor generated from GAC patient (right). B. Representative co-immunofluorescent staining images of GRK3 and SOX9 expression on more PC specimen. Scale bars, 25µm. C. Representative co-immunofluorescent staining images of GRK3 and SOX9 expression on paired human primary tumor and PC samples. Scale bars, 25µm.  Supplementary Figure 2. Overexpression of GRK3 in GAC cells increased malignant behaviors of GAC. A. Cell viability assay (MTS) was performed in MKN45-GRK3 and MKN45-GFP control cells. Fold increases of cell growth in day 6 and day 8 in media with the indicated FBS % was plotted on y-axis. **P&lt;0.001.  B. Tumor sphere formation assay was performed in MKN45 GFP and GRK3 OE cells (n = 3 biological replicates). *P&lt;0.01. C. Invasion assay was performed in KATO III GFP, GRK3 OE and GRK3 Mut cells (n = 3 biological replicates) as described in Materias&amp;Methods. *P&lt;0.01; **P&lt;0.001. Scale bar: 25µm. D. Colony formation assay was performed in KATO III GFP, GRK3 OE and GRK3 Mut cells (n = 3 biological replicates) as described in Materias&amp;Methods. Scale bar: 100µm. **P&lt;0.01; ***P&lt;0.001. E. GFP and GRK3 OE cells of KATO III was cultured in 96-well plate, and MTS assay was performed at Day 3, 5, 7 and 9. **P&lt;0.01; ***P&lt;0.001. F. Tumor sphere assay was performed in NC and GRK KD cells of GA0518. G. AGS cells seeded in a 96-well plate were infected with a series of shRNA of GRK3 and scramble virus doses, from 20ul to 2.5ul per well. Cell survival and proliferation were measured by alamar blue viability assay 7 days after infection. X-axis represents the series of shRNA virus doses in the infections. Values on Y-axis are relative growth which was normalized to the scramble control shRNA for each virus dose for either cell line. P values were calculated using 2-sided Student’s t-test. ***: P &lt; 0.005; *: P &lt; 0.05. Supplementary Figure 3. Flow charts of our compound library screening for identifying GRK3 inhibitors A. Schematic illustration of our compound library screening to identify candidates of GRK3 inhibitors. B. Detailed flow chart of the library screening, using Z'-LYTE Kinase Assay (the Ser/Thr 16 Peptide Kit from Life Technology). GRK3 recombinant protein was first mixed with diluted compound library, followed by the addition of ATP, peptide substrate and Z-LYTE buffer. Effective GRK3 inhibitors were identified when they blocked GRK3-mediated phosphorylation of the peptide substrates. Unphosphorylated peptides were cleaved after the addition of Developing Reagent, which resulted in no FRET (Fluorescence Resonance Energy Transfer). Supplementary Figure 4. LD2 specifically inhibited the proliferation of GRK3 OE cells in MKN45 and decreased colony formation and invasion in AGS cells. A. Cell viability assay was performed in MKN45 GFP and GRK3 OE cells treated with indicated concertation of LD2. *P&lt;0.01; **P&lt;0.001. B. Colony formation assay was performed in AGS treated with indicated concertation of LD2. *P&lt;0.01; **P&lt;0.001. C. Invasion assay was performed in AGS treated with indicated concertation of LD2. **P&lt;0.001. Supplementary Figure 5. KD YAP1 in GRK3 OE GAC cells rescue the malignant phenotype induced by GRK3 OE in both KATO III and MKN45 cells. A&amp;B. Reduced YAP1 in GRK3 OE cells suppress tumor cell invasion induced by GRK3 OE in KATO III and MKN45 cells. C&amp;D. Knockdown YAP1 in GRK3 OE cells suppress tumor sphere formation induced by GRK3 OE in KATO III and MKN45 cells. Supplementary Figure 6. LD2 inhibited YAP expression and YAP/TEAD transcriptional activity. A. Representative co-immunofluorescent staining images of GRK3 and YAP expression on GA0518 cell treated with or without LD2 at 0.5µM for 24 hours. Scale bars, 25µm. B. YAP1 promoter activity was determined at 48 hours after co-transfection of YAP1-luciferase promoter and Renilla luciferase and then treated with LD2 at indicated dosage (n = 3 biological replicates). **P&lt;0.001. C. YAP1/TEAD transcriptional activity was detected by cotransfection of Gal4-Tead and 5XUAS-luciferase plasmids with YAP1 cDNA into GA0518 cells and then treated with LD2 at indicated dosage. Luciferase reporter activity was measured after 48 hours. (n = 3 biological replicates).  (** P &lt; 0.01). Supplementary Figure 7. Increased expression of YAP1 targets-CTGF, Cyr61 and Survivin (Birc5) in GRK3 OE cells compared to normal control cells (NC) in both MKN45 and KATOIII cells. A. CTGF and GRK3 expression in NC and GRK3 OE cells in both MKN45 and KATO III cell line, examined by immunofluorescence. B. Survivin expression in NC and GRK3 OE cells in both MKN45 and KATO III cell line, examined by immunofluorescence. C. Cyr61 expression in NC and GRK3 OE cells in both MKN45 and KATO III cell line, examined by immunofluorescence. Scale bar: 50 µm. Supplementary Figure 8. GRK3 inhibitor LD2 inhibited stem cell trait of GAC. A. Tumor sphere formation assay was performed in GA0518, GA0804 and AGS cells treated with indicated concentration of LD2 (n = 3 biological replicates). *P&lt;0.01, **P&lt;0.001, ***P&lt;0.0001. Scale bar 100µm. B. ALDH1A1+ population was labeled using the ALDEFLUOR kit in GA0804 and AGS cells as described in Materials&amp; Methods. Supplementary Figure 9. Effects of GRK3 OE on peritoneal metastases, while LD2 decrease peritoneal metastases induced by GRK3 OE in PC metastatic model. A. Peritoneal metastasis were shown in the indicated group. B&amp;C. Representative bioluminescence images and quantification of the mice in the indicated group. P=0.045; P=0.0079.