Supplemental Methods, Supplemental Figure 1-24 from Noninvasive Measurement of mTORC1 Signaling with <sup>89</sup>Zr-Transferrin
Authors: Truillet Charles , Cunningham John T. , Parker Matthew F.L. , Huynh Loc T. , Conn Crystal S. , Ruggero Davide , Lewis Jason S. , Evans Michael J. 
Published: April 2023

<p>Supplemental Methods, Supplementary Figure 1. A. Real time PCR data showing the suppression of PTEN mRNA levels by an anti PTEN shRNA in GBM and PCa cell lines. The suppression of PTEN mRNA was statistically significant (P < 0.05). B. Immunoblot data showing suppression of PTEN protein in the cell line cohort. Supplementary Figure 2. A. Real time PCR data showing the upregulation of PIK3CA wild type and mutant mRNA in HEK293 cells. Lysates were collected for analysis 48 hours after transfection. The upregulation was statistically significant (P < 0.05). SSupplementary Figure 3. A. Real time PCR data showing the upregulation of mTOR wild type and mutant mRNA in HEK293 cells. Lysates were collected for analysis 48 hours after transfection. The upregulation was statistically significant compared to mock (P < 0.05). Supplemental Figure 4. Right: In vitro uptake data showing that transient knockdown of TSC1 or TSC2 results in higher intracellular uptake of 125I-transferrin in NIH-3T3 cells. The cells were transfected for 48 hours prior to running the uptake assay for 1 hour. Left: In vitro uptake data showing that 125I-transferrin is internalized into TSC1-/- and TSC2-/- MEFs to a greater extent than the respective wild type subline. The uptake assay was conducted for 1 hour. The uptake was statistically significant compare to the respective parental line (P < 0.05). Supplemental Figure 5. Real time PCR data showing the knockdown of TSC1 or 2 mRNA by siRNA. Dharmacon smartPOOL siRNAs were transfected into NIH-3T3 cells for 48 hours prior to harvest. The gene knockdown was statistically significant (P < 0.01). Supplemental Figure 6. A photograph and PET images of wild type and PTEN null prostate tissues show higher uptake of the radiotracer in the PTEN null tissues. The contralateral lobes of the prostate are arranged with bilateral symmetry along a vertical axis cutting through the middle of the petri dish. Additional normal tissues from the urogenital tract are shown for comparison. Abbreviations: AP = anterior prostate, DP = dorsal prostate, VP = ventral prostate, LP = lateral prostate, B = bladder, SV = seminal vesicle, Ur = urethra. Supplemental Figure 7. A summary of the biodistribution data for all tissues in the Pb-cre PTENlox/lox mice. The statistics for the prostate lobes were described in the text. Supplementary Figure 8. Immunoblot data showing changes in phosphoprotein abundance in the PI3K/Akt/mTOR signaling axis, indicative of bioactivity for the respective kinase inhibitor in PTEN null GBM and PCa cell lines. Note no change in phosphoprotein levels for doxorubicin, as expected. Lysates were harvested 6 hours after the initiation of therapy. Supplementary Figure 9. A summary of the 125I-transferrin uptake data for all doses and treatment durations of BEZ235 in the PTEN null cell lines. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05). Supplementary Figure 10. A summary of the 125I-transferrin uptake data for all doses and treatment durations of INK128 in the PTEN null cell lines. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05). Supplementary Figure 11. A summary of the 125I-transferrin uptake data for all doses and treatment durations of RAD001 in the PTEN null cell lines. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05). Supplementary Figure 12. A summary of the 125I-transferrin uptake data for all doses and treatment durations of doxorubicin in the PTEN null cell lines. Supplementary Figure 13. Immunoblot data showing changes in phosphor-S6 (Ser 240/44), indicative of bioactivity for the respective kinase inhibitor in T47D or HCT115 cells. Note no change in phosphoprotein levels for doxorubicin, as expected. Lysates were harvested 24 hours after the initiation of therapy. Supplementary Figure 14. A summary of the 125I-transferrin uptake data for all doses and treatment durations of BEZ235, INK128, RAD001 and doxorubicin in the T47D cells. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05 for all treatments except doxorubicin). Supplementary Figure 15. A summary of the 125I-transferrin uptake data for all doses and treatment durations of BEZ235, INK128, RAD001 and doxorubicin in the HCT115 cells. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05 for all treatments except doxorubicin). Supplementary Figure 16. Real time PCR data showing the efficacy of siRNA knockdown using the Dharmacon smartPOOL reagents. These data were collected 48 hours post transfection. The knockdown was statistically significant compared to cells treated with non-targeting (NT) siRNA (P < 0.01). Supplementary Figure 17. Pharmacological inhibition of mTORC1 activity suppresses TFRC transcription in vitro. (Left). Real time PCR data collected in vitro after 48 hours of treatment shows a substantial reduction in TFRC mRNA by treatment with targeted therapies, and no effect of doxorubicin, as expected, in U87MG, LNCaP-AR and PC3 cells. The doses of drugs were BEZ235 (100 nM), INK128 (100 nM), RAD001 (10 nM) and Doxorubicin (500 nM). (Right). An in vitro binding assay with 125I-DF1513 (a monoclonal antibody recognizing an extracellular epitope on human TFRC) shows that the abundance of cell surface TFRC is reduced by BEZ235 (100 nM), INK128 (100 nM), and RAD001 (10 nM) compared to vehicle. No effect was observed with Doxorubicin (500 nM). Cells were treated for 48 hours with drug, and incubated with 125I-DF1513 for 30 min at 4o C before isolating and counting the cell associated activity. In all cases, treatment with kinase inhibitors resulted in statistically significant decreases compared to vehicle and doxorubicin (P < 0.01). Supplementary Figure 18. Genetic inhibition of mTORC1 activity suppresses TFRC transcription in vitro. ( Left).Real time PCR data collected in vitro after 48 hours of transfection shows a substantial reduction in TFRC mRNA induced by siRNA to mTOR and RAPTOR, but not the mTORC2 component PRR5. Knockdown was confirmed with rtPCR. (Right) An in vitro binding assay with 125I-DF1513 shows that the abundance of cell surface TFRC is reduced by siRNAs (100 nmol) targeting mTOR and RPTOR compared to a nontargeting (NT) siRNA. No effect was observed with siRNA targeting PRR5, as expected. Cells were transfected for 48 hours, and incubated with 125I-DF1513 for 30 min at 4o C before isolating and counting the cell associated activity. In all cases, siRNA to mTOR or RPTOR resulted in statistically significant decrease in TFRC mRNA or protein compare to NT and siRNA against PRR5 (P < 0.01). Supplemental Figure 19. A summary of the biodistribution data for mice bearing subcutaneous U87 tumors, and treated with BEZ235, INK128, RAD001, or doxorubicin. The statistics for the tumor associated values were described in the text. Supplementary Figure 20. ROI analysis of the U87MG tumors in mice treated with vehicle or the indicated inhibitor. This data was derived from the same cohort used to generate the biodistribution data. The changes associated with the kinase inhibitors were statistically significant compare to vehicle and doxorubicin (P < 0.01). Supplementary Figure 21. A summary of the biodistribution data for mice bearing subcutaneous LNCaP tumors, and treated with BEZ235, INK128, RAD001, or doxorubicin. The statistics associated with the tumor values were described in the text. Supplementary Figure 22. A summary of the biodistribution data for mice bearing subcutaneous PC3 tumors, and treated with BEZ235, INK128, RAD001, or doxorubicin. The statistics associated with the tumor values were described in the text. Supplemental Figure 23. In vitro uptake data showing the impact of treatment dosing and changing exposure time in LNCaP-AR cells. The values on the X-axis refer to the amount of time LNCaP-AR was exposed to drug prior to 1 hour incubation with 125I-Tf. The cell surface and internalized activity was quantified, and normalized to vehicle treated cells. All treatments with the exception of ARN (1 ï�­M) and ARN + RAD were statistically significant compared to vehicle (P < 0.01). Supplemental Figure 24. Biodistribution data from mouse tissues and tumors treated with vehicle, RAD001, ARN-509, or the combination for two days prior to injection with 89Zr-Tf. The biodistribution was collected 48 hours post injection of radiotracer. The statistics associated with the tumor values were described in the text.</p>