(A) Dose–response curves of CAMA-1, MCF7, and T47D everolimus sensitive and resistant cells under everolimus treatment. Cells were treated with increasing concentration of everolimus for 4 days and …
Dose–response curves of CAMA-1, MCF7, and T47D everolimus sensitive and resistant cells under ONC201/TIC10 treatment. Cells were treated with increasing concentration of ONC201/TIC10 for 4 days and …
(A) Representative images of spheroid growth of sensitive (Venus, green) or resistant (mCherry, red) cells cultured in everolimus, ONC201/TIC10, or combination treated media at the indicated …
(A) 3D cultures of primary patient-derived ER+ BC cells from ascites or pleural effusion treated with everolimus, ONC201/TIC10, or combination at the indicated concentrations for 4 days. Cell …
Representative images of spheroid growth of primary patient-derived ER+ BC cells collected during everolimus treatment or post-everolimus and therapy resistant, cultured and treated with everolimus, …
(A) CAMA-1, MCF7, T47D everolimus sensitive and resistant cells were treated for 24 hr with ONC201/TIC10 at the indicated concentrations, and cell lysates were immunoblotted for TUFM, TFAM, BiP, …
Original files of the full blot images for Figure 4A.
Original files of the full blot images for Figure 4B.
Figures with uncropped blot images for Figure 4A, B.
CAMA-1, MCF7, T47D everolimus sensitive and resistant cells were treated for 24 hr with everolimus, ONC201/TIC10, and combination at the indicated concentrations. Cell lysates were immunoblotted for …
Original files of the full blot images for Figure 4—figure supplement 1.
Figures with uncropped blot images for Figure 4—figure supplement 1.
Scatter plots displaying the enrichment scores (Y-axis) of (A) REACTOME cell cycle signature, (B) REACTOME ATF4 activated genes in response to endoplasmic reticulum stress signature, (C) REACTOME …
Cells were treated for 18 hr with indicated concentrations of everolimus, ONC201/TIC10, and combination. Mitochondrial respiration was measured using Seahorse XF Cell Mito Stress assay and oxygen …
Cells were cultured for 18 hr. Mitochondrial respiration was measured using Seahorse XF Cell Mito Stress assay and oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) graphs …
Cells were treated for 18 hr with indicated concentrations of ONC201/TIC10 and everolimus. Mitochondrial respiration was measured using Seahorse XF Cell Mito Stress assay and oxygen consumption …
Patient diagnosis | Sample | Therapy lines | Drugs | Therapy lines post-everolimus | |
---|---|---|---|---|---|
P1 | Metastatic ER+/HER2− | Ascites | 6 | 12 | 2 |
P2 | Metastatic ER+/PR+/HER2− | Ascites | 7 | 9 | 2 |
P3 | Metastatic ER+/PR+/HER2− | Pleural effusion | 7 | 9 | 1 |
P4 | Metastatic ER+/PR+/HER2− | Ascites | 10 | 14 | 5 |
P5 | Metastatic ER+/PR+/HER2− | Ascites | 5 | 6 | 1 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (Homo-sapiens) female | CAMA-1 Breast; Mammary gland: adenocarcinoma | ATCC | HTB-21 | |
Cell line (Homo-sapiens) female | MCF7 Breast; Mammary gland: adenocarcinoma epithelial cell | ATCC | HTB-22 | |
Cell line (Homo-sapiens) female | T47D Breast; Mammary gland: carcinoma ductal epithelial cell | ATCC | HTB-133 | |
Cell line (Homo-sapiens) female | Primary, P1, P2, P3, P4, P5 | This paper. City of Hope IRB #07047 and #17334 | P1, P2, P3, P4, P5 | Pleural effusion or ascites samples from patients |
Transfected construct (mammalian) | LeGO-V2 (Venus) | Weber et al., 2008 Addgene #27340 | Addgene #27340; RRID:Addgene_27340 | Lentiviral construct to transfect and express Venus fluorescent protein |
Transfected construct (mammalian) | LeGO-C2 (mCherry) | Weber et al., 2008 Addgene #27339 | Addgene #27339; RRID:Addgene_27339 | Lentiviral construct to transfect and express mCherry fluorescent protein |
Antibody | anti-BiP (C50B12) (rabbit monoclonal) | Cell Signaling Technology | Cat#3177; RRID:AB_2119845 | WB (1:1000) |
Antibody | anti-pFoxO3a (Ser294) (rabbit polyclonal) | Cell Signaling Technology | Cat#5538; RRID:AB_10696878 | WB (1:1000) |
Antibody | anti-FoxO3a (D19A7) (rabbit monoclonal) | Cell Signaling Technology | Cat#12829; RRID:AB_2636990 | WB (1:1000) |
Antibody | anti-pERK1/2 Thr202/Tyr204 (197G2) (rabbit monoclonal) | Cell Signaling Technology | Cat#4377; RRID:AB_331775 | WB (1:1000) |
Antibody | anti-ERK1/2 (137F5) (rabbit monoclonal) | Cell Signaling Technology | Cat#4695; RRID:AB_390779 | WB (1:1000) |
Antibody | anti-pAKT Ser473 (D9E) (rabbit monoclonal) | Cell Signaling Technology | Cat#4060; RRID:AB_2315049 | WB (1:1000) |
Antibody | anti-AKT (C67E7) (rabbit monoclonal) | Cell Signaling Technology | Cat#4691; RRID:AB_915783 | WB (1:1000) |
Antibody | anti-pS6 Ser240/244 Ribosomal Protein (D68F8) (rabbit monoclonal) | Cell Signaling Technology | Cat#5364; RRID:AB_10694233 | WB (1:1000) |
Antibody | anti-S6 Ribosomal Protein (54D2) (mouse monoclonal) | Cell Signaling Technology | Cat#2317; RRID:AB_2238583 | WB (1:1000) |
Antibody | anti-β-actin (8H10D10) (mouse monoclonal) (HRP Conjugate) | Cell Signaling Technology | Cat#12262; RRID:AB_2566811 | WB (1:2000) |
Antibody | anti-β-actin (13E5) (rabbit monoclonal) (HRP Conjugate) | Cell Signaling Technology | Cat#5125; RRID:AB_1903890 | WB (1:2000) |
Antibody | anti-rabbit IgG, HRP-linked (goat polyclonal) | Cell Signaling Technology | Cat#7074; RRID:AB_2099233 | WB (1:2000) |
Antibody | anti-mouse IgG, HRP-linked (horse polyclonal) | Cell Signaling Technology | Cat#7076; RRID:AB_330924 | WB (1:2000) |
Antibody | anti-CREB-2/ATF-4 (B-3) (mouse monoclonal) | Santa Cruz Biotechnology | Cat#390063; RRID:AB_2810998 | WB (1:300) |
Antibody | anti-mtTFA (TFAM) (C-9) (mouse monoclonal) | Santa Cruz Biotechnology | Cat#376672; RRID:AB_11150497 | WB (1:200) |
Antibody | anti-TRAIL (55B709.3) (mouse monoclonal) | Thermo Fisher Scientific | Cat# MA1-41027; RRID:AB_1087999 | WB (1.5 μg/ml) |
Antibody | anti-CHOP (rabbit polyclonal) | Proteintech | Cat#15204-1-AP; RRID:AB_2292610 | WB (1:1000) |
Antibody | anti-TUFM (rabbit polyclonal) | Thermo Fisher Scientific | Cat#PA5-27511; RRID:AB_2544987 | WB (1:500) |
Antibody | anti-Total OXPHOS (mouse monoclonal) | Abcam | Cat#ab110411; RRID:AB_2756818 | WB (1:1000) |
Chemical compound, drug | Everolimus (RAD001) | Selleckchem | Cat#S1120 | Dissolved in DMSO |
Chemical compound, drug | ONC201/TIC10 | Selleckchem | Cat#S7963 | Dissolved in DMSO |
Commercial assay or kit | CellTiter-Glo Luminescent Cell Viability Assay | Promega | Cat#G7573 | |
Commercial assay or kit | CellTiter-Glo 3D Cell Viability Assay | Promega | Cat#G9682 | |
Commercial assay or kit | EasySep CD45 Depletion Kit II | StemCell Technologies | Cat#17898 | |
Commercial assay or kit | EasySep Dead Cell Removal (Annexin V) Kit | StemCell Technologies | Cat#17899 | |
Commercial assay or kit | Agilent Seahorse XF Cell Mito Stress Test Kit | Agilent Technologies | Cat#103015-100 | |
Software | GraphPad Prism software | GraphPad (https://graphpad.com) | RRID:SCR_002798 | Version 9.3.1 |
Software | Gen5 software | Biotek Instruments (https://www.agilent.com/) | Version 3.05 | |
Software | Seahorse Wave Desktop Software 2.6 | Agilent Technologies (https://www.agilent.com/) | RRID:SCR_014526 | Version 2.6 |
Software | ImageJ software | ImageJ (http://imagej.nih.gov/ij/) | RRID:SCR_003070 |