Journal: Nature Communications

Article Title: The PIK3CA/AKT pathway drives therapy resistance in rhabdomyosarcoma

doi: 10.1038/s41467-025-66632-9

Figure Lengend Snippet: A Histopathology of xenografted tumors. Representative RD parental sensitive (S1) and resistant models (R1, R4) showing co-immunofluorescence staining of PI3Kα (Alexa Fluor 546) and MDR1 (Alexa Fluor 488); IHC for p-AKT (Ser473) and p-NRF2 (Ser40; A , left). Quantification of percent positive cells within each model mean +/− ST noted on images or in graphical analysis (right). N = 3 in sensitive models and n = 6 in resistant models (biological replicates, RD and Rh41). Data are mean ± SD. B Western blot analysis of RD and Rh41 cell line grown after selection in xenografted mice. Data shown are from one representative experiment and was repeated three times using biological replicates with similar results. The lysates derive from the same experiment but were run on different gels. One gel was used for analysis of PI3K p110, Nrf2, p-Nrf2, AKT, p-AKT, p-4EBP1, S6, another for EGFR, PI3K p85, p-PI3K p85, MDR1, MRP1, BCRP, p-PTEN, PTEN, 4EBP1, and p-S6. Both gels were processed in parallel. C Quantification of cell viability following drug treatment in RD and Rh41 models. Cells treated for 4 days. Grey denotes comparisons that were not done. Scale (1.0 growth/viable and 0.0 dead). Data shown as mean from three independent biological replicates. D Representative confocal image of RD therapy sensitive and resistant model following treatment with PARPi-FL (red) and temozolomide (TMZ) or in combination with alpelisib PIK3CAα inhibitor (OT + AL) or Tariquidar ABC transport inhibitor (OT + TA). Samples were counterstained with NucBlue. Z-stack image (top, D ) and cell cross-section (bottom, D ). E Quantification of relative PARPi-FL uptake in therapy sensitive and OT resistant RD and Rh41 cells subjected to OT, OT + alpelisib (AL), OT + tariquidar (TA). n > 248 cells per condition. Shown is a representative example from one of three independent, biological replicate experiments; similar results observed across all experiments ( D , E ). F Representative images and G flow cytometry analysis of RD parental sensitive (S1) and resistant cells (R1, R4) showing fluorescence eFluxx-ID® signals after treating with DMSO or alpelisib PIK3CAα inhibitor (AL). The fluorescence signal of the dye negatively correlates with the activity of the ABC transporters. Images are representative of three independent biological replicates ( F , G ). P < 0.05 was considered statistically significant. ANOVA followed by Dunnett post hoc test was used to make comparisons between sensitive and resistant clones ( A ). ANOVA followed by two-sided Student’s T -test comparisons within each resistant model ( E ). Scale bar equals 25 µm ( A ), 10 µm ( D , upper; F ), and 2 µm ( D , lower).

Article Snippet: Primary antibodies used included PTEN (CST, 9559), p-PTEN (Ser380/Thr382/383) (CST, 9551), EGFR (abcam, ab52894), PI3K p85 (CST, 4292), p-PI3K p85 (Tyr458) (CST, 17466), AKT (CST, 4691), p-AKT (Ser473) (CST, 4060), NRF2 (abcam, ab62352), p-NRF2 (Ser40) (abcam, ab76026), S6 (CST, 2317), p-S6 (Ser235/236) (CST, 81736), 4EBP1 (CST, 9644), p-4EBP1 (Thr37/46) (CST, 2855), MDR1/ABCB1 (CST, 13342), MRP1/ABCC1 (CST, 72202), BCRP/ABCG2 (CST, 42078), GAPDH (CST, 5174).

Techniques: Histopathology, Immunofluorescence, Staining, Western Blot, Selection, Flow Cytometry, Fluorescence, Activity Assay, Clone Assay