Journal: Clinical Cancer Research

Article Title: PI3Kα Inhibitor and Degrader Inavolisib Can Co-opt FGFR2 to Enhance Responses in Patients with PIK3CA -Mutated Solid Tumors and in Preclinical Models

doi: 10.1158/1078-0432.CCR-25-1459

Figure Lengend Snippet: Inavolisib sensitivity depends on high FGFR2 expression. A, Legend related to panels in B–I . B, Cell lines were treated with 0.03 μmol/L of the FGFR2i or 2-μmol/L lapatinib for 1 hour followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). C, Cell lines were treated with inavolisib or the FGFR2i at various concentrations for 1 hour. Cell lysates were immunoprecipitated with an antibody against p85β, followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). IB, immunoblotting. D, Following RAS-GTP pulldown, cell lines were treated with the FGFR2i or lapatinib for different durations and immunoblotted with the antibodies indicated at left. Representative results from experiments ( n = 2). E, Cell lysates from cells treated with inavolisib alone or in combination with the FGFR2i or lapatinib for 4 hours were immunoprecipitated with RAS antibody and blotted with p110α antibody. F, Mechanistic model of the effects of FGFR2 and HER2 inhibition on HER3 and RAS activity. FGFR2-high–expressing cell lines induced PI3K signaling through both HER3 and WT RAS activity (top) compared with HER2-induced PI3K signaling through HER3 but not RAS activity (bottom). G, FGFR2-high–expressing cell lines, MFM223 and SUM52PE, were treated with inavolisib, FGFR2i, or lapatinib for 1 hour. Membrane fractions were analyzed by reciprocal co-IP with one another using HER3 or FGFR2 antibody and Western blotting with FGFR2, HER3, RAS, and p85β antibody. Representative results from experiments ( n = 2). H, SUM52PE, MFM223, and MFE280 cells were treated with inavolisib single-agent or in combination with the FGFR2i or lapatinib for 6 hours. Ubiquitinated proteins were pulled down from the membrane fraction with TUBE1 reagent and blotted with p110α antibody. Representative results from experiments ( n = 2). I, Western blots of the inhibitor response in PI3K signaling (pHER3 and pAKT) in PIK3CA mutant MFM223 and PIK3CA WT SUM52PE; cell lines were treated with 0.5-μmol/L inavolisib or 1-μmol/L alpelisib for different durations. Representative results from experiments ( n = 2). J, Ratio of inavolisib and alpelisib GR 50 values in FGFR2-high ( n = 12) vs. FGFR2-low ( n = 9) expressing cell lines harboring PIK3CA mutations, as assessed in a 5-day viability assay. Data are represented as median (center line) ± IQR (25th to 75th percentile, box) and ± full range (minimum to maximum, whiskers). P value was calculated using Wilcoxon rank-sum test. Representative results from experiments ( n = 2). WB, Western blotting.

Article Snippet: Antibodies to p110α (cat. No. 4249, RRID: AB_2165248), pAKT Ser473 (cat. No. 4060, RRID: AB_2315049), pS6 S235/236 (cat. No. 2211, RRID: AB_331679), HER3 (cat. No. 12708, RRID: AB_2721919), HER2 (cat. No. 2242, RRID: AB_331015), pHER2 Y1221/Y1222 (cat. No. 2243, RRID: AB_490899), pHER3 Y128 (cat. No. 4791, RRID: AB_2099709), pHER3 Y1328 (cat. No. 14525, RRID: AB_2798501), pPLCγ Y783 (cat. No. 14008, RRID: AB_2728690), pERK T202/T204 (cat. No. 9101, RRID: AB_331646), p4EBP T37/46 (cat. No. 9459, RRID: AB_330985), FGFR1 (cat. No. 9740, RRID: AB_11178519), FGFR2 (cat. No. 11835, RRID: AB_2797742), FGFR3 (cat. No. 4574, RRID: AB_2246903), FGFR4 (cat. No. 8562, RRID: AB_10891199), pFGFR Y653/654 (cat. No. 3476, RRID: AB_331369), and pFRS2A Y196 (cat. No. 3864, RRID: AB_2106222) were obtained from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Immunoprecipitation, Inhibition, Activity Assay, Membrane, Co-Immunoprecipitation Assay, Mutagenesis, Viability Assay

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Effects of different valine concentrations on the phosphorylation of downstream proteins in the mTOR signaling pathway. ( A ) Western blotting was used to detect the protein expression levels of phosphorylated mTOR (P-mTOR) and phosphorylated S6K1 (P-S6K1). ( B ) The phosphorylation level of mTOR protein (P-mTOR/mTOR) was quantified and calculated using β-actin and mTOR as internal references. ( C ) The phosphorylation level of S6K1 protein (P-S6K1/S6K1) was quantified and calculated using β-actin and S6K1 as internal references. ( D ) Western blotting was used to detect the protein expression levels of phosphorylated 4EBP1 (P-4EBP1) and phosphorylated RPS6 (P-RPS6). ( E ) The phosphorylation level of 4EBP1 protein (P-4EBP1/4EBP1) was quantified and calculated using β-actin and 4EBP1 as internal references. ( F ) The phosphorylation level of RPS6 protein (P-RPS6/RPS6) was quantified and calculated using β-actin and RPS6 as internal references. ( G ) Western blotting was used to detect the protein expression levels of phosphorylated eEF2 (P-eEF2). ( H ) The phosphorylation level of eEF2 protein (P-eEF2/eEF2) was quantified and calculated using β-actin and eEF2 as internal references. The error bars represent the SD ( n = 3). ANOVA was used to detect the differences between groups, and the LSD method was combined for uncorrected exploratory pairwise comparisons, while the Duncan method was used for conservative multiple comparison corrections. Different lowercase letters indicate significant differences ( p < 0.05), while different uppercase letters indicate significant differences ( p < 0.01).

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: Phospho-proteomics, Western Blot, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Effects of valine and rapamycin on the phosphorylation levels of mTOR downstream proteins. ( A ) The phosphorylation levels of mTOR (P-mTOR) in MAC-T cells treated with varying concentrations of rapamycin were assessed using Western blotting. ( B ) The P-mTOR level (P-mTOR/mTOR) in MAC-T cells treated with 100 nM rapamycin was quantified and calculated using β-actin and mTOR as internal references. ( C ) Western blotting was used to detect the protein expression levels of P-mTOR and P-S6K1. ( D ) The P-mTOR level (P-mTOR/mTOR) was quantified and calculated using β-actin and mTOR as internal references. ( E ) The P-S6K1 level (P-S6K1/S6K1) was quantified and calculated using β-actin and S6K1 as internal references. ( F ) Western blotting was used to detect the protein expression levels of P-4EBP1 and P-RPS6. ( G ) The P-4EBP1 level (P-4EBP1/4EBP1) was quantified and calculated using β-actin and 4EBP1 as internal references. ( H ) The P-RPS6 level (P-RPS6/RPS6) was quantified and calculated using β-actin and RPS6 as internal references. ( I ) Western blotting was used to detect the protein expression levels of P-eEF2 and α-casein. ( J ) The P-eEF2 level (P-eEF2/eEF2) was quantified and calculated using β-actin and eEF2 as internal references. ( K ) The α-casein protein expression level was quantified using β-actin as an internal reference. The error bars represent the SD ( n = 3). ANOVA was used to detect the differences between groups, and the LSD method was combined for uncorrected exploratory pairwise comparisons, while the Duncan method was used for conservative multiple comparison corrections. Different lowercase letters indicate significant differences ( p < 0.05), while different uppercase letters indicate significant differences ( p < 0.01).

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: Phospho-proteomics, Western Blot, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Primer sequences of internal reference genes and target genes.

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Detailed information of antibodies.

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: