Alexa fluor 647 conjugate

Antibodies against CHOP (L63F7, mAb#2895), phospho‐eIF2α ((Ser51) D9G8, mAb#3398), eIF2α (#9722), ATF4 (D4B8, mAb#11815), BiP (#3183), HSP90 (E289, #4875), PARP (46D11, mAb#9532), IL‐1β (D3U3E, mAb#12703), phospho‐p38 MAPK (D3F9, mAb#4511), p38 MAPK (D13E1, mAb#8690), GAPDH (14C10, mAb#2118) and EpCAM (VU1D9, mAb#5447, Alexa Fluor^® 647 Conjugate) were purchased from Cell Signaling. Anti‐phospho‐IRE1α (Ser724, PA1‐16927) antibody was purchased from Thermo Scientific Inc Anti‐CPT1A (8F6AE9, ab128568), anti‐LXRα ([PPZ0412]‐ChIP Grade (ab41902)) and anti‐GFP (Ab5450) antibodies were purchased from Abcam. Clarity Western ECL substrate was purchased from Bio‐Rad.

Cell cycle analysis was performed with the Click‐iT Plus EdU Flow Cytometry Assay Kit (Invitrogen) and DAPI as DNA staining reagent (BD Biosciences). Five days after shRNA transduction, cells were incubated with 10 μM EdU solution for 1 h, washed with 3 ml 1% BSA/DPBS, fixed with 4% PFA, permeabilized, and incubated with EdU detection solution and DAPI. Stained cells were acquired on a BD LSR II flow cytometer (BD Biosciences) within 1 h.
The effects of verteporfin on apoptotic and mitotic rates were assessed by flow cytometric detection of cleaved PARP and phosphorylated histone H3^S10, respectively. MLS cells grown in medium supplemented with 2% FBS were treated with 0.25 μM verteporfin for 72 h, detached using 0.025% trypsin (Life Technologies), fixed in 2% PFA, washed in PBS, and permeabilized in 0.25% Triton X‐100/PBS for 5 min on ice. After an additional washing step, cells were stained for 60 min with PE Mouse anti‐Cleaved PARP (Asp214) (BD Biosciences) and phospho‐Histone H3^S10 (#9716, Alexa Fluor 647 Conjugate, Cell Signaling) antibodies. Fluorescence intensity was measured using a FACSCanto II flow cytometer, and data were analyzed using the FACSDiva software (BD Biosciences).

To determine the relative expression of NIS proteins exclusively on the extracellular membrane of the cell, 1.5x10⁵ HeLa and HeLa-HA-NIS cells, in triplicate, were fixed in 500 μl 4% paraformaldehyde in PBS, then washed twice with 3 ml FACS buffer. Cells were stained with HA-tag (6E2) mouse mAb-Alexa Fluor 647 Conjugate (Cell Signaling Technology, Danvers, MA, #3444) 1:40 dilution for 1 hour at room temperature in 100 μl FACS buffer. Cells were washed twice with 3 ml cold FACS buffer. Singlets were isolated and gated on non-transduced HeLa cells incubated with antibody.

Cells were first fixed in 4% paraformaldehyde for 10 min and then permeabilized with 0.2% TritonX-100 in PBS for 10 min before being blocked with PBS containing 5% bovine serum albumin (BSA) for 30 min. Thereafter, the cells were sequentially incubated with anti-PHB2 and anti-NPM primary antibodies and then with anti-rabbit IgG (H + L) (Alexa Fluor^® 647 Conjugate, Cat # 4414, Cell Signaling Technology) and anti-mouse IgG (H + L) (Alexa Fluor^® 488 Conjugate, Cat # 4408, Cell Signaling Technology), respectively. The images were acquired using an Olympus FV-1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).

Cells were deposited on cytospins, fixed and permeabilized in methanol (−20 °C, 15 min), blocked (BlockAid; Thermo Fisher Scientific), and rabbit anti-LC3A/B mAb Alexa Fluor 647 Conjugate (13394, 1:50, Cell Signaling Technology) in PBS + 1% BSA + 0.3% Triton X-100 was applied overnight (4 °C), followed by 300 nM DAPI (AnaSpec, 5 min). After mounting (Prolong Diamond Antifade, Thermo Fisher Scientific) and drying overnight (4 °C), images were acquired using a Leica TCS SP5 II confocal laser scanning microscope (HCX PL APO CS 63×/1.4N oil immersion objective) and LAS-AF lite 2.0 (Leica Microsystems).

MOLM-16 and OCI-AML3 cells were treated for 6 h with AZD1208 (2 μM), AZD2014 (1 μM), or the combination. Cells were then fixed with 1.6% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and subjected to permeabilization in ice-cold methanol (70% in PBS; 1 mL/million cells) for 20 min. After washing twice, cells were resuspended in 1% bovine serum albumin in PBS. Antibodies were added to the cell suspension and incubated for 30 min. Antibodies used were Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) mouse monoclonal antibody (Alexa Fluor 488 Conjugate #4374; Cell Signaling Technology, Beverly, MA); Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP rabbit monoclonal antibody (Alexa Fluor 647 Conjugate #4851; Cell Signaling Technology); Phospho-Akt (Ser473) rabbit monoclonal antibody (Alexa Fluor 647 Conjugate #A88881; Beckman Coulter, Inc., Brea, CA); and CXCR4 (CD184-APC, #555976; BD Biosciences). After washing twice, cells were resuspended and analyzed by a Gallios flow cytometer (Beckman-Coulter).

EGFR expression of the cells used in this study was measured by Western blotting and flow cytometry.
Whole-cell lysates were collected and Western blotting was performed as described previously18 (link). Primary antibodies and the titres used in this study were as follows: rabbit monoclonal anti-EGFR antibody (D38B1, #4267, Cell Signaling Technology, Inc., Danvers, MA, USA; 1:1,000), rabbit monoclonal anti-β-Actin antibody (13E5, #5125, Cell Signaling; 1:5,000). β-Actin served as loading controls for whole-cell lysates.
EGFR expression on the cell surface was detected with flow cytometry by measuring the fluorointensity of dye-conjugated anti-EGFR antibody which binds to cell-surface EGFR with a minor modification from a previous report38 (link). In brief, cells were washed twice with PBS, and they were stained for 30 minutes on ice with AlexaFluor 647-conjugated anti-EGFR or control antibodies in 2% FBS in PBS. Then, cells were washed twice with PBS and analysed with a flow cytometer (BD LSRFortessa Analyzer; BD Biosciences, San Jose, CA, USA). Antibodies and the titres were as follows: AlexaFluor 647-conjugated monoclonal anti-EGFR antibody (D38B1, #5588, Cell Signaling; 1:50), rabbit IgG isotype control (Alexa Fluor 647 Conjugate) antibody (#3452, Cell Signaling; 1:50). Collected data were analysed using FlowJo X software (Tree Star, Inc., Ashland, OR, USA).