Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: Related to . A, To interrogate the Cancer Genome Atlas (TCGA) database using online Kaplan-Meier plotter tools to evaluate the correlation between the expression of SRC mRNA and overall survival of various solid tumor patients. For each analysis, patients were splitted by median. The number of patients in each category and the Hazard Ratio are shown in the graph. B, To analyze the mRNA levels of SRC in UCEC, LIHC, KIRC, KIRP and BRCA patients from TCGA by using the GEPIA online database. C, To analyze the spheres formation of pancreatic PDAC ASPC-1 cells by the treatment of combined silencing SRC with Dasatinib (50nM) or separately Dasatinib (50nM). (n ≥ 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001). D-F, To analyze the inhibitory effects on the Dasatinib resistance cell proliferation, metastasis and stemness of silencing SRC by MTT assay (D), transwell assay (E) and spheres formation assay respectively. (n ≥ 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001). G, To detect the cellular localization of Nc-SRC in the ASPC-1 surviving cells after the Dasatinib treatment for 24h by using immunofluorescence assay. (n≥ 3 independent experiments). H , To detect the cellular localization of phosphorylation inactivated mutant SRC (Y419F) in HEK293T cells after the transfection of SRC (Y419F) for 24h by using immunofluorescence assay. (n≥ 3 independent experiments).

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Expressing, MTT Assay, Transwell Assay, Tube Formation Assay, Immunofluorescence, Phospho-proteomics, Mutagenesis, Transfection

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: A, Schematic diagrams of screening the key protein-protein interactions that enriched in the surviving cell nucleus after Dasatinib treatment. First, 21 potential enriched nuclear genes were obtained from the up-regulated genes (Fc>2, p <0.05) in the surviving cells after SRC kinase inhibitor treatment (GSE81235, GSE166617) and SRC kinase inhibitor resistant cells (GSE129071) and nuclear genes from the online GSEA database. The patients from TCGA database were divided into two groups. The correlations between the above 21 genes and the overall survival of LIHC, KIRP, KIRC, PDAC and BRCA patients were analyzed through the online Kaplan-Meier plotter database. Then, 10 oncogenes were selected. B, The SRC-CoIP mass spectrometry data show that TRIB3 interacted with SRC in surviving cells after Dasatinib treatment for 24h. C, The Real-time PCR results show that the TRIB3 mRNA level was increased in surviving cells after Dasatinib treatment for 24h. (n = 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001) D, The western blotting data show higher TRIB3 expression in the PDAC cell ASPC-1, LIHC cell HUH7 and KIRP cell 769-P. (n = 3 independent experiments) E-F, The western blotting data show the accumulation of TRIB3 and SRC in ASPC-1, HUH7 and 769-P cells after Dasatinib treatment for 24h (E) and in the Dasatinib-resistant ASPC-1-DasaRes and HUH7-DasaRes cells (F). (n = 3 independent experiments). G-H, The enrichment of TRIB3/SRC interaction in ASPC-1, HUH7 and 769-P cells determined by CoIP (G) and Duolink PLA assay (H). (n = 3 independent experiments)

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Protein-Protein interactions, Mass Spectrometry, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: A, Schematic diagrams of screening the direct target gene of Nc-SRC. First, 128 potential SRC target genes were obtained from the differentially expressed genes (Fold-change, Fc>1.5 and Fc<0.66, p <0.05) in mammary epithelial cells from spontaneous breast cancer PyMT mice with mammary-gland-specific knockout of SRC (GSE224876) and SRC-predicted genes from the hTFtarget database. The patients from the TCGA database were divided into two groups. The correlations between the above 128 genes and the overall survival of LIHC, KIRP, KIRC, PDAC and BRCA patients were analyzed through the online Kaplan-Meier plotter database. Then, 13 oncogenes and 11 anti-oncogenes were selected. Next, the SRC target genes were determined by Real-time PCR assay, luciferase reporter assay and CHIP-PCR assay. B-C, The Real-time PCR results show that silencing SRC decreased the mRNA levels of 6 oncogenes (B) and increased that of 1 anti-oncogene (C) in ASPC-1 cells. (n = 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001) D, The Real-time PCR data indicate that 3 oncogenes were enriched in surviving cells after Dasatinib (50nM) treatment for 24h in ASPC-1 cells. (n = 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001) E, The western blotting data show the decreased phosphorylation of mutant SRC (Y419F) and increased phosphorylation of SRC (Y419D). (n = 3 independent experiments) F, The Real-time PCR data indicate that 2 oncogenes were increased after the transfection of SRC (Y419F) for 36h in HEK293T cells. (n = 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001) G, The luciferase reporter data show that SRC (Y419F) promoted the transcription of SPC24 and BIRC5 . (n = 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001) H, The binding region of SRC-WT and SRC (Y419F) to the SPC24 promoter determined by the CHIP-PCR assay. (n = 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001)

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Western Blot, Phospho-proteomics, Mutagenesis, Transfection, Binding Assay

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: Related to . A, Silencing Nc-SRC decreased SPC24 expression in ASPC-1-Resis and HUH7-Resis cells . B-C, The spheres formation assay (C) data show that silencing Nc-SRC exhibited stronger inhibition as compared to silencing SPC24 (B) in Dasatinib resistance ASPC-1-DasaRes cells. (n = 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001)

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Expressing, Tube Formation Assay, Inhibition

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: A-B, Silencing both TRIB3 and SRC shows stronger inhibition of cell proliferation and migration compared to separately silencing TRIB3 or SRC in ASPC-1, 769-P, and HUH7 cells, as determined by MTT assay (A) and Transwell assay (B). (n ≥ 3 independent experiments; Data are presented as mean ± SEM; t test, * P <0.05, *** P <0.001, **** P <0.0001). C, Silencing TRIB3 or SRC reduced the sphere-formation ability, silencing combined TRIB3 and SRC is shown more reduction as compared with separately silencing TRIB3 in above four type cells. (n ≥ 3 independent experiments; Data are presented as mean ± SEM; t test, * P <0.05, *** P <0.001, **** P <0.0001).

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Inhibition, Migration, MTT Assay, Transwell Assay

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: A, The western blotting data show that silencing TRIB3 expression reduced SRC accumulation in ASPC-1, HUH7, 769-P cells. (n = 3 independent experiments) B, The Real-time PCR results show that there is no significant inhibition in SRC mRNA levels after silencing TRIB3 in ASPC-1, 769-P, and HUH7 cells. (n = 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001) C, HEK293T cells were transfected with CTRL-HA or TRIB3-HA vector for 24h, and then treated with cycloheximide (CHX, 20 μM) for the indicated time. The western blotting data show that TRIB3 extended the half-life of SRC protein degradation. D, The ASPC-1 cells were transfected with CTRL-siRNA or SRC-siRNA for 24h, and then treated with proteasome inhibitor MG132 (10μM) or autophagy inhibitor bafilomycin BAF (200nM) for 8h. The western blotting data show that silencing TRIB3 reduced SRC expression in ASPC-1 cells, which was inhibited by MG132 but not by BAF. (n = 3 independent experiments) E, The western blotting data show that silencing CHIP (gene name STUB1) extended the half-life of SRC degradation from 5.9 h to more than 24 h. The ASPC-1 cells were transfected with CTRL-siRNA or CHIP-siRNA for 24h, then treated with CHX (20 μM) for the indicated time. (n = 3 independent experiments) F, HEK293T cells were transfected with the indicated plasmid for 24h. The western blotting data show that overexpression of CHIP-GFP decreases SRC protein expression, but simultaneous overexpression of TRIB3 reverses this function. (n = 3 independent experiments) G, The TRIB3/SRC interaction intercepted the CHIP/SRC interaction. HEK293T cells were transfected with the indicated plasmid for 24h. The cell extracts were IP with an anti-SRC-Myc Ab and blotted with an anti-CHIP-GFP or anti-TRIB3-HA Ab. (n = 3 independent experiments) H, The effect of TRIB3 on SRC ubiquitylation by CHIP. HEK293T cells were transfected with the indicated plasmid. His-ubiquitin-conjugated proteins were pulled down with Ni-NTA-agarose from cell lysates. Total protein lysates and Ni-NTA-agarose eluates were immunoblotted and stained for FLAG, His, HA, and Myc. (n ≥ 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001)

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Inhibition, Transfection, Plasmid Preparation, Over Expression, Ubiquitin Proteomics, Staining

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: Related to . A, The western blotting data show that silencing TRIB3 expression reduced SRC accumulation in MCF7 cells. (n = 3 independent experiments) B, To predict the E3 ubiquitin ligase of SRC by using the online Unibrowser 2.0 database, 7 known E3 ligase were followed study. C, SRC CoIP mass spectrometry assay results show that SRC interaction with above 7 E3 ligase in wild type ASPC-1 cells (C0), but only with FBXL7 and STUB1 in surviving cells after Dasatinib treatment for 24h (C24). D, The second-order spectrum of FBXL7 and STUB1 from SRC CoIP mass spectrometry of C24 cells. E, CHIP interaction with active p-SRC(Y416) and inactive p-SRC(Y530). HEK293T cells were transfected with CHIP-GFP and SRC-Myc plasmid for 24h, the cell extracts were IP with an anti-CHIP-GFP Ab and blotted with an anti-CHIP-GFP, anti-SRC-Myc, anti-p-SRC(Y416) and anti-p-SRC(Y530) Ab. (n = 3 independent experiments) F, CHIP interaction with active SRC(Y416D) and inactive SRC(Y416F). HEK293T cells were transfected with indicated plasmid for 24h, the cell extracts were IP with an anti-SRC-Myc Ab and blotted with an anti-CHIP-GFP, anti-SRC-Myc, anti-p-SRC(Y416). (n = 3 independent experiments) G, Inactive SRC(Y416F) decreased myristoylation of SRC. HEK293T cells were transfected with indicated plasmid for 24h, the cell extracts were IP with an anti-SRC-Myc Ab and blotted with an anti-SRC-Myc and anti-myristoylation Ab. (n = 3 independent experiments). H, The western blotting data show that silencing CHIP extended the half-life of TRIB3 degradation from 1.84 h to more than 8 h. The ASPC-1 cells were transfected with CTRL-siRNA or CHIP-siRNA for 24h, then treated with CHX (20 μM) for the indicated time. (n = 3 independent experiments)

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Mass Spectrometry, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: A, Silencing the whole SRC enhanced TRIB3 accumulation in ASPC-1, HUH7, and 769-P cells, as determined by Western blotting assay. B-D, Overexpression of inactive mutant SRC (Y419F) enhanced TRIB3 protein expression (B), mRNA levels (C) and transcription (D), as determined by the western blotting, real-time PCR and promoter-luciferase reporter assay in HEK293T cells. (n = 3 independent experiments) E, The SRC-WT and SRC(Y419F) proteins direct bind to the TRIB3 promoter sequence. HEK293T cells were transfected with the indicated plasmid, and ChIP analysis was used to monitor the binding of SRC-WT or SRC(Y419F) to the TRIB3 promoter. Data represent at least three separate experiments and are shown as the mean ± SEM., t test; t test, *P<0.05, ***P<0.001, ****P<0.0001. F-H, Overexpression of STAT1 reversed the TRIB3 protein (F), mRNA (G) and transcription (H) accumulation in surviving cells after Dasatinib treatment. HEK293T cells were transfected with the indicated plasmid for 36h, and treated with Dasatinib for 24h. Western blotting, Real-time PCR or luciferase reporter analysis was used to monitor the expression of the indicated proteins, mRNA or promoter activation. Data represent at least three separate experiments. I, The STAT1-Flag protein directly binds to the TRIB3 promoter sequence. HEK293T cells were transfected with STAT1-Flag plasmid, and ChIP analysis was used to monitor the binding of STAT1-Flag to the TRIB3 promoter. Data represent at least three separate experiments and are shown as the mean ± SEM., t test; t test, *P<0.05, ***P<0.001, ****P<0.0001.

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Western Blot, Over Expression, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Sequencing, Transfection, Plasmid Preparation, Binding Assay, Activation Assay

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: Related to . A, Twenty-three potential TRIB3 transcription factor that interacted with SRC were gained by using online hTFtarget database, NCBI database and SRC CoIP mass spectrometry data. B, 13 of above transcription factors were confirmed by JASPAR database. C, 6 of above transcription factors were known phosphorylated by active SRC. D, Silencing TRIB3 or SPC24 reduced the spheres formation ability, silencing combined TRIB3 and SPC24 showed similar reduction as compared with silencing SRC in ASPC-1 DasRes and HUH7 DasRes cells. (n ≥ 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001).

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Mass Spectrometry

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: A-B, Mapping the SRC regions binding to TRIB3. Schematic diagram of full-length SRC and deletion mutants. C-E , TRIB3 interacted with the region from residues 439 to 469 in the kinase domain of SRC, which is away from the ATP binding site of the 276-284aa, and 298aa regions determined by the UniProt database. HEK293T cells were transfected with indicated plasmid. Cell extracts were IP with an anti-HA Ab and blotted with an anti-TRIB3-HA or anti-SRC-Myc Ab. Data represent at least three separate experiments. F, The kinetic interaction between the α-helical peptide S1-2 and the TRIB3 protein was determined by BLI assay. Data represent at least three separate experiments . G, TS1-2 interrupted the SRC/TRIB3 interaction. The fused peptide TS1-2 was designed by linking a cell-penetrating peptide TAT (GRKKRRQRRR) to S1-2 using a glycine–glycine (GG) linker. HEK293T cells were treated with TAT (10 μM) or TS1-2 (10 μM) for 12h. Cell extracts were IP with an anti-HA Ab and blotted with an anti-TRIB3-HA or anti-SRC-Myc Ab. Data represent at least three separate experiments . H, TS1-2 simultaneously reduced both the TRIB3 and SRC expression determined by Western blotting. Data represent at least three separate experiments . I-J, TS1-2 accelerated TRIB3 degradation from 2.3 to 0.3 h (I) and SRC degradation from 6.8 to 2 h (J) in ASPC-1 cells. The ASPC-1 cells were transfected with TAT (10 μM) or TS1-2 (10 μM), and treated with CHX (20 μM) for the indicated time. Cell extracts were blotted with an anti-TRIB3 or anti-SRC Ab. (Data are shown as the mean ± SEM; n ≥ 3 independent experiments; Half-life t 1/2 were calculated by GraphPad 9.0 software)

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Software

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: A-C, TS1-2 significantly reduced cell proliferation, migration, and stemness compared to Dasatinib. The combination of with TS1-2 and Dasatinib showed excellent synergistic effects in ASPC-1, HUH7 and 769-P cells, as determined by MTT assay (A), Transwell assay (B), and mammospheres formation assay (C). (n ≥ 3 independent experiments; Data are presented as mean ± SEM; t test, *P<0.05, ***P<0.001, ****P<0.0001).

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Migration, MTT Assay, Transwell Assay, Tube Formation Assay

Journal: bioRxiv

Article Title: Targeting Non-catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

doi: 10.1101/2025.02.15.638389

Figure Lengend Snippet: A, Schematic diagrams of the ASPC-1 xenograft BALB/c Nude mice model and the treatment of TS1-2 and/or Dasatinib at indicated time. B-D, TS1-2 significantly reduced the tumor volume (B-C) and tumor weight (D). The combination of TS1-2 and Dasatinib showed excellent synergistic effects in ASPC-1 xenograft BALB/c Nude mice. (n=5, Data are presented as mean ± SEM; One-way ANOVA (B), Unpaired t test (D), *P<0.05, ***P<0.001, ****P<0.0001). E, Tissue extracts were obtained by ultrasonic lysis of tumor tissue from ASPC-1 animals, and blotted with the indicated Ab. (n ≥ 3 independent experiments from different animal). F, Schematic diagrams of the 4T1 xenograft BALB/c mice model and the treatment of TS1-2 and/or Dasatinib at indicated time. G-I, TS1-2 significantly reduced the tumor volume (G-H) and tumor weight (I). The combination of TS1-2 and Dasatinib showed excellent synergistic effects in 4T1 xenograft BALB/c mice. (n=8, Data are presented as mean ± SEM; One-way ANOVA (B), Unpaired t test (D), *P<0.05, ***P<0.001, ****P<0.0001). I, Tissue extracts were obtained by ultrasonic lysis of tumor tissue from 4T1 (C) animals, and blotted with the indicated Ab. (n ≥ 3 independent experiments from different animals)

Article Snippet: ASPC-1, 769-P or HUH7 cells were pretreated with indicated siRNAs for 36 h, 10 μM TAT or TS1-2 for 24 h, or indicated concentrations of Dasatinib (MCE) for 24 h. For mammosphere assays, ASPC-1 cells, 769-P cells or HUH7 cells (500 cells/well) were seeded on 96-well ultralow attachment plates (Corning, 3474) in StemXVivo Serum-Free Tumorsphere Media (R&D, CCM012).

Techniques: Lysis