Figures S7 A and S7B for statistical graph). (C) Dose-response curves and corresponding IC50 of sunitinib in ABATsh LYN Y397F and ABATsh SYK Y323/525F ccRCC (786-O and 768-P) cells and ABATsh SYK Y323/525F ccRCC cells treated with R406 and ABATsh LYN Y397F ccRCC cells treated with PD173952. x axis, log10 drug concentrations. y axis, Activity%, the percentage of cellular or biological activity in the drug-treated group relative to the control group. (D) Tube formation assays of HUVECs cultured in conditioned medium from ABATsh SYK Y323/525F , ABATsh SYK WT , ABATsh treated with R406, ABATsh LYN Y397F , ABATsh LYN WT , or ABATsh treated with PD173952 ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see
Figures S7 C and S7D for statistical graph). (E) Chick embryo chorioallantoic membrane (CAM) assays with conditioned medium from ABATsh SYK Y323/525F , ABATsh SYK WT , ABATsh treated with R406, ABATsh LYN Y397F , ABATsh LYN WT , and ABATsh treated with PD173952 ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see
Figures S7 E and S7F for statistical graph). (F) Compared to those in the monotherapy with sunitinib, there were fewer EdU-positive cells in the combination therapy of sunitinib with R406 or PD173952 in ABATsh SYK Y323/525F or ABATsh LYN Y397F ccRCC cells. (G and H) Mutual interaction between SYK phosphorylation and LYN phosphorylation. Representative blots ( n = 3 experiments) are shown (see
Figures S7 G and S7H for statistical graph). (I) Treatments were combined with vehicle (vegetable oil), sunitinib (40 mg/kg in vegetable oil), R406 (5 mg/kg PD173952 in vegetable oil), a combination of sunitinib and R406 (40 mg/kg sunitinib and 5 mg/kg PD173952 in vegetable oil), a combination of sunitinib and PD173952 (40 mg/kg sunitinib and 20 mg/kg PD173952 in vegetable oil), or PD173952 (20 mg/kg PD173952 in vegetable oil) separately in heterograft tumors (769-P S, 769-P R cells), and tumor growth was recorded ( n = 4/group) (see
Figures S7 I and S7J for statistical graph). The data are presented as the means ± SEMs. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test, two-way ANOVA with Tukey’s multiple comparisons test, or unpaired t test where appropriate. Each spot represents one subject. ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. See also
Figure S7 . " width="100%" height="100%">
Journal: iScience
Article Title: Decoding sunitinib resistance in ccRCC: Metabolic-reprogramming-induced ABAT and GABAergic system shifts
doi: 10.1016/j.isci.2024.110415
Figure Lengend Snippet: Inhibiting LYN or SYK enhances sunitinib efficacy against resistant ccRCC (A and B) Decreased SYK and LYN phosphorylation in ABAT-knockdown ccRCC cells (786-O and 769-P) (see Figures S7 A and S7B for statistical graph). (C) Dose-response curves and corresponding IC50 of sunitinib in ABATsh LYN Y397F and ABATsh SYK Y323/525F ccRCC (786-O and 768-P) cells and ABATsh SYK Y323/525F ccRCC cells treated with R406 and ABATsh LYN Y397F ccRCC cells treated with PD173952. x axis, log10 drug concentrations. y axis, Activity%, the percentage of cellular or biological activity in the drug-treated group relative to the control group. (D) Tube formation assays of HUVECs cultured in conditioned medium from ABATsh SYK Y323/525F , ABATsh SYK WT , ABATsh treated with R406, ABATsh LYN Y397F , ABATsh LYN WT , or ABATsh treated with PD173952 ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see Figures S7 C and S7D for statistical graph). (E) Chick embryo chorioallantoic membrane (CAM) assays with conditioned medium from ABATsh SYK Y323/525F , ABATsh SYK WT , ABATsh treated with R406, ABATsh LYN Y397F , ABATsh LYN WT , and ABATsh treated with PD173952 ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see Figures S7 E and S7F for statistical graph). (F) Compared to those in the monotherapy with sunitinib, there were fewer EdU-positive cells in the combination therapy of sunitinib with R406 or PD173952 in ABATsh SYK Y323/525F or ABATsh LYN Y397F ccRCC cells. (G and H) Mutual interaction between SYK phosphorylation and LYN phosphorylation. Representative blots ( n = 3 experiments) are shown (see Figures S7 G and S7H for statistical graph). (I) Treatments were combined with vehicle (vegetable oil), sunitinib (40 mg/kg in vegetable oil), R406 (5 mg/kg PD173952 in vegetable oil), a combination of sunitinib and R406 (40 mg/kg sunitinib and 5 mg/kg PD173952 in vegetable oil), a combination of sunitinib and PD173952 (40 mg/kg sunitinib and 20 mg/kg PD173952 in vegetable oil), or PD173952 (20 mg/kg PD173952 in vegetable oil) separately in heterograft tumors (769-P S, 769-P R cells), and tumor growth was recorded ( n = 4/group) (see Figures S7 I and S7J for statistical graph). The data are presented as the means ± SEMs. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test, two-way ANOVA with Tukey’s multiple comparisons test, or unpaired t test where appropriate. Each spot represents one subject. ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. See also Figure S7 .
Article Snippet: PD173952 , MedChemExpress , CAS: 305820-75-1.
Techniques: Phospho-proteomics, Knockdown, Activity Assay, Control, Cell Culture, Membrane
Figure S8 A for statistical graph). Representative blots ( n = 3 experiments) are shown. (E) Western blot of key genes enriched in the pathway comprising LYN Y397E or LYN Y397F in ccRCC cells (786-O and 769-P) (see
Figure S8 B for statistical graph). Representative blots ( n = 3 experiments) are shown. (F) Western blot of key genes enriched in the pathway in SYK Y323/525E or SYK Y323/525F ccRCC cells (786-O and 769-P) (see
Figure S8 C for statistical graph). The data represent one of two independent experiments. (G) Western blot of key genes enriched in the pathway in SYK Y323/525E ccRCC cells (786-O and 769-P) treated with R406 (see
Figure S8 D for statistical graph). Representative blots ( n = 3 experiments) are shown. The data are presented as the means ± SEMs. Groups were compared using two-way ANOVA with Tukey’s multiple comparisons test. Each spot represents one subject. POS, positive control spot; ns, not significant; ∗∗∗∗ p < 0.0001. See also
Figure S8 . " width="100%" height="100%">
Journal: iScience
Article Title: Decoding sunitinib resistance in ccRCC: Metabolic-reprogramming-induced ABAT and GABAergic system shifts
doi: 10.1016/j.isci.2024.110415
Figure Lengend Snippet: Elevated phosphorylation levels of SYK and LYN activate the Akt and STAT pathways (A and B) Differential gene expression analysis of wild-type ccRCC cells and stable SYK- and LYN-phosphorylated ccRCC cell activation by sequencing. (C) KEGG enrichment analysis of the DEGs. (D) Western blot of key genes enriched in the pathway in LYN Y397E ccRCC cells (786-O and 769-P) treated with PD173952 (see Figure S8 A for statistical graph). Representative blots ( n = 3 experiments) are shown. (E) Western blot of key genes enriched in the pathway comprising LYN Y397E or LYN Y397F in ccRCC cells (786-O and 769-P) (see Figure S8 B for statistical graph). Representative blots ( n = 3 experiments) are shown. (F) Western blot of key genes enriched in the pathway in SYK Y323/525E or SYK Y323/525F ccRCC cells (786-O and 769-P) (see Figure S8 C for statistical graph). The data represent one of two independent experiments. (G) Western blot of key genes enriched in the pathway in SYK Y323/525E ccRCC cells (786-O and 769-P) treated with R406 (see Figure S8 D for statistical graph). Representative blots ( n = 3 experiments) are shown. The data are presented as the means ± SEMs. Groups were compared using two-way ANOVA with Tukey’s multiple comparisons test. Each spot represents one subject. POS, positive control spot; ns, not significant; ∗∗∗∗ p < 0.0001. See also Figure S8 .
Article Snippet: PD173952 , MedChemExpress , CAS: 305820-75-1.
Techniques: Phospho-proteomics, Gene Expression, Activation Assay, Sequencing, Western Blot, Positive Control
Figure S9 A for statistical graph). The data represent one of two independent experiments. (E) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in ccRCC cells (786-O and 769-P) treated with R406 (see
Figure S9 B for statistical graph). The data represent one of two independent experiments. (F) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in LYN Y397E or LYN Y397F ccRCC cells (786-O and 769-P) (see
Figure S9 C for statistical graph). The data represent one of two independent experiments. (G) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in SYK Y323/525E or SYK Y323/525F ccRCC cells (786-O and 769-P) (see
Figure S9 D for statistical graph). The data represent one of two independent experiments. (H and I) Inhibited EREG or PGF in ABAT-knockdown ccRCC cells (786-O and 769-P) (see
Figures S10 A and S10B for statistical graph). (J) Tube formation assays using HUVECs cultured in CM from ABATsh PGFsi, ABATsh EREGsi, and ABATsh NCsi ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see
Figures S10 C and S10D for statistical graph). (K) Chick embryo chorioallantoic membrane (CAM) assays with conditioned medium from ABATsh PGFsi, ABATsh EREGsi, and ABATsh NCsi ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see
Figures S10 E–S10G for statistical graph). The data are presented as the means ± SEMs. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test, two-way ANOVA with Tukey’s multiple comparisons test, or unpaired t test where appropriate. Each spot represents one subject. NC, negative control. ∗∗ p < 0.01, ∗∗∗ p < 0.0001, ∗∗∗∗ p < 0.0001. See also
Figures S9 and . " width="100%" height="100%">
Journal: iScience
Article Title: Decoding sunitinib resistance in ccRCC: Metabolic-reprogramming-induced ABAT and GABAergic system shifts
doi: 10.1016/j.isci.2024.110415
Figure Lengend Snippet: Elevated phosphorylation levels of SYK and LYN upregulate PGF to promote angiogenesis (A–C) The intersection of angiogenesis genes and key genes enriched in the pathway (A). (B) A protein-protein interaction (PPI) network of the top 15 key genes from (A) was constructed. Partial vascular-related key genes (C). (D) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in ccRCC cells (786-O and 769-P) treated with PD173952 (see Figure S9 A for statistical graph). The data represent one of two independent experiments. (E) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in ccRCC cells (786-O and 769-P) treated with R406 (see Figure S9 B for statistical graph). The data represent one of two independent experiments. (F) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in LYN Y397E or LYN Y397F ccRCC cells (786-O and 769-P) (see Figure S9 C for statistical graph). The data represent one of two independent experiments. (G) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in SYK Y323/525E or SYK Y323/525F ccRCC cells (786-O and 769-P) (see Figure S9 D for statistical graph). The data represent one of two independent experiments. (H and I) Inhibited EREG or PGF in ABAT-knockdown ccRCC cells (786-O and 769-P) (see Figures S10 A and S10B for statistical graph). (J) Tube formation assays using HUVECs cultured in CM from ABATsh PGFsi, ABATsh EREGsi, and ABATsh NCsi ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see Figures S10 C and S10D for statistical graph). (K) Chick embryo chorioallantoic membrane (CAM) assays with conditioned medium from ABATsh PGFsi, ABATsh EREGsi, and ABATsh NCsi ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see Figures S10 E–S10G for statistical graph). The data are presented as the means ± SEMs. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test, two-way ANOVA with Tukey’s multiple comparisons test, or unpaired t test where appropriate. Each spot represents one subject. NC, negative control. ∗∗ p < 0.01, ∗∗∗ p < 0.0001, ∗∗∗∗ p < 0.0001. See also Figures S9 and .
Article Snippet: PD173952 , MedChemExpress , CAS: 305820-75-1.
Techniques: Phospho-proteomics, Construct, Western Blot, Knockdown, Cell Culture, Membrane, Negative Control
