Mtor 7c10

CSCs were washed twice with PBS and then lysed in RIPA lysis buffer (#R0278, Sigma) (150 mM NaCl, 1.0% IGEPAL^® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, and Proteinase Inhibitor 1×). Post sonication, cell lysates were centrifuged at 14,000 g for 10 min at 4 °C, and the supernatants were used for western blotting. The lysates were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and then stained with 0.1% Ponceau S solution (#P3504, Sigma) to ensure equal loading of the samples. After being blocked with 5% non-fat milk for 60 min, the membranes were incubated with primary antibodies mTOR (7C10) (1:1000, #2983, Cell Signaling, Danvers, MA, USA); AMPKα (1:1000, #2532, Cell Signaling); acetyl-CoA carboxylase (C83B10) (1:1000, #3676, Cell Signaling); fatty acid synthase (C20G5) (1:1000, #3180, Cell Signaling); pyruvate dehydrogenase (C54G1) (1:1000, #3205, Cell Signaling); GLUT4 (1:2500, #ab65267, Abcam, Cambridge, UK); BAD (1:500, #sc-8044, SantaCruz, TX, USA); and β-Actin (13E5) (1:1000, #4970, Cell Signaling) overnight at 4 °C, and the bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies using the ECL Western Blotting Substrate (GE #RPN2106, Amersham, Pittsburgh, PA, USA) on a Chemi-Doc (Bio-Rad, Hercules, CA, USA) imaging analyser.

After the total proteins of cells in each group were isolated with total protein extraction kit (Transgen, DE101-01), protein samples were treated with SDS-PAGE protein loading buffer (Beyotime, P0015). Then the cell proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, which were transferred to a polyvinylidene fluoride membrane (Millipore), washed with TBST buffer (Solarbio, T1081). The membrane was sealed with 5% skim milk for 1h, and incubated with primary antibody at 4 °C overnight, and the second antibody incubated at 37 °C for 1h. Then, the samples were washed 3 times with TBST buffer (Solarbio, T1081), and ECL luminescence solution (Biosharp, BL520A) was added. The protein bands were observed using a chemiluminescence gel imaging system (Bio-RAD, USA). The used antibodies are as follows: GAPDH (Transgen, HC301-01), HSPA5 (Proteintect, 11587-1-AP), LC3B (Abcam, ab192890), P62 (BOSTER, M00300-1), Phospho-mTOR (Ser2448) (D9C2) (Cell Signaling Technology, 5536T), mTOR (7C10) (Cell Signaling Technology, 2983T), Phospho-Akt (Ser473) (D9E) (Cell Signaling Technology, 4060S), Akt (pan) (C67E7) (Cell Signaling Technology, 4691S), Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (ab275018), Goat Anti-mouse IgG (Transgen, HS201-01), Goat anti-rabbit IgG (Transgen, HS101-01).

DOTAP and 1,2-distearoryl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol-2000) ammonium salt (DSPE-PEG₂₀₀₀) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). DSPE-PEG-anisamide was synthesized in our lab as described previously68 (link). DeadEnd Fluorometric TUNEL assay kits and Luciferase Assay System assay substrates were obtained from Promega (Madison, WI). Linear PEI hydrochloride with average molecular weight 8,000, dicyandiamide, HA and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Rabbit monoclonal antibodies Phospho-AMPKα(Thr172) (40H9) (catalogue # 2535), AMPKα (D5A2) (catalogue # 5831), Phospho-mTOR (Ser2448) (D9C2) XP (catalogue # 5536), mTOR (7C10) (catalogue # 2983), LC3b (D11) XP (catalogue # 3868), GAPDH (14C10) (catalogue # 2118) and Anti-rabbit IgG, horseradish peroxidase-linked Antibody (catalogue # 7074) were purchased from Cell Signaling Technology (Beverly, MA). BCL2 siRNA (target sequence: 5′-AACAUCGCCCUGUGGAUGACU-3′), VEGF siRNA (target sequence: 5′-ACCUCACCAAGGCCAGCAC-3′) and control siRNA (target sequence: 5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Sigma-Aldrich.

Immunofluorescence and co-localization analysis were used in Fig 5j and 5k. Cells were plated onto poly-L-lysine-coated confocal dishes. After treatments, cells were fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) at room temperature for 10 min and washed/permeabilized with 1× PBST solution (1× PBS and 0.1% Triton X-100) for another 15 min. Then, the cells were blocked with blocking solution (1× PBS with 1% BSA) at room temperature for 30 min. Staining was performed with the indicated primary antibodies in blocking solution (1:200 dilution) at 4°C overnight. The cells were stained with cross-adsorbed secondary fluorescent antibodies (1:100 in blocking solution). For the merged images, Elab Fluor 594 (E-AB-1059, Elabscience, for anti-LAMP2 IFs) and Elab Fluor 488 (E-AB-1055, Elabscience, for anti-mTOR IFs) are shown in magenta and cyan, respectively. Imaging was performed with a 100× oil immersion objective on Olympus confocal microscope. The antibodies used included LAMP2 (66301-1-Ig, Proteintech), mTOR (7C10) (2983, Cell Signaling).
To quantify the co-localization of mTOR with the lysosomal marker LAMP2, the Fiji software was used. Forty individual cells from independent fields were selected for the analysis. The Coloc2 plugin was used to calculate the Pearson’s correlation coefficient.

Tumour tissues were harvested immediately after sacrificing the NSG mice, fixed with 10% neutral buffered formalin for 18–24 h, processed and embedded in paraffin. The samples were sectioned at 6 µm using microtome (RM2135, Leica, USA). H&E staining was performed following standard protocols. For IHC, antigen retrieval was carried out by boiling the sections in citrate buffer (pH 6) for 10 min, followed by cooling at RT for 1 h. To eliminate endogenous peroxidases, tissues were treated in methanol containing 3% H₂O₂ for 30 min. Tissues were permeabilized with .1% Triton X‐100 for 30 min. Sections were further blocked in 10% goat serum for 30 min followed by incubation with primary antibodies at 4°C overnight. Tissues were washed with PBS and subsequently incubated with secondary antibody at 37°C for 1 h. Antibodies against Ki67 (#16667, Abcam), HES1 (D6P2U; #11988), Phospho‐p44/42 MAPK (ERK 1/2; #4370S) and mTOR (7C10; #2983) were purchased from Cell Signaling Technology, USA. Stained slides were imaged under a bright‐field microscope (EVOS XL Core, Invitrogen). For IHC scoring, both intensity and percentage of positive cells were considered. A total of 10 microscopy fields were reviewed in each section. Tumour cells with brown cytoplasm and/or nucleus or membrane were considered positive. Percentage of stained tumour cells is represented as IHC staining score.

Primary antibodies used in this study were anti-KRT17 (D7327, Cell Signaling Technology, Danvers, MA, USA), KRT17 (EPR1624Y, Abcam, Cambridge, UK), KRT17 (E2, Dako, Glostrup, Denmark), KRT5 (EP1601Y, Abcam), KRT6 (EPR1603Y, Abcam), KRT16 (EP1615Y Abcam), mTOR (Y391, Abcam), mTOR (7C10, Cell Signaling Technology), 4E-BP1 (53H11, Cell Signaling Technology), phospho-4E-BP1(Thr37/46)(236B4, Cell Signaling Technology), AKT1 (Y89, Abcam), AKT1 (phospho S473)(EP2109Y, Abcam), GAPDH (D16H11, Cell Signaling Technology), Histone H3 (D1H2, Cell Signaling Technology), Beta-Tubulin (9F3, Cell Signaling Technology), 14-3-3 sigma (1.N.6, Abcam), Glut1 (EPR3915, Abcam), Ki67 (SP6, Abcam), Digoxigenin (Roche Diagnostics, Basel, Switzerland), Flag M2 (Sigma-Aldrich, St. Louis, MO, USA), HA (12CA5, Roche Diagnostics) antibodies. Secondary antibodies used in this study were Envision/HRP (Dako), HRP-donkey anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA), HRP-rabbit anti-mouse IgG (Thermo Fisher Scientific), Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific), Alexa Fluor 594 goat anti-rabbit IgG (Thermo Fisher Scientific) and Alexa Fluor 488 rabbit anti-mouse IgG (Thermo Fisher Scientific).