Alexa fluro 594

All chemicals were purchased from Sigma-Aldrich unless noted otherwise. Antibodies against c-myc, cleaved caspase3, cleaved PARP, and GST were purchased from Cell Signaling Technology (Beverly, MA). Anti-GAPDH, γ-H2A.X, β-actin, CDK5, p35, GFP, HA, p-S/T/Y, and Thiophosphate ester antibodies were from Abcam (Cambridge, MA). Anti-CLIC4 antibody was from Novagen (Madison, WI). Fluorescent anti-mouse or anti-rabbit IgG antibodies conjugated with Alexa Fluro 488 or Alexa Fluro 594 were from Invitrogen. Protein G Dynabeads were from Life technologies, and Glutathione Sepharose beads were from GE. ATP-γ-S and p-nitrobenzyl mesylate (PNBM) were from Abcam.

Freshly excised liver tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich, #441244), dehydrated and embedded in paraffin, or directly snap frozen in Tissue-Tek™ O.C.T. compound (Sakura Finetek™ 4583) for cryosectioning. H&E staining was performed on 5 or 8 µm paraffin sections. Immunostaining was performed on 8 μm frozen sections. First, samples were fixed in ice-cold 4% paraformaldehyde for 15 min and washed 3 times in PBS/0.1% Triton X-100 buffer at room temperature. Then the slides were incubated with 10% normal goat serum/0.3 M glycine for 1 h to block non-specific binding, followed by overnight incubation with primary antibodies at 4 °C. After rinsing 3 times with 0.1% Triton X-100 in PBS for 15 min, samples were incubated with secondary antibodies conjugated to Alexa Fluor-488 (Invitrogen, A-11055) or Alexa Fluro 594 (Invitrogen, A-21442) at 25 °C for 2 h, followed by PBS washing. Samples were counterstained with Hoechst and finally mounted using VECTASHIELD Antifade mounting medium (Vector Laboratories, #H-1000). Primary antibodies used were rabbit polyclonal anti-hFIX/PTC (Abcam, #ab97619) at dilution of 1:200, and goat polyclonal anti-Albumin (Bethyl, #A90-134A) at dilution of 1:300. The secondary antibodies were diluted at 1:500.

The frozen primary or PDX breast cancer tissues were fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, blocked with 3% BSA, and stained with an anti-TRIB3 (1:100, Abcam, ab137526), anti-CD68 (1:100, Abcam, ab955), anti-FOXO1 (1:100, Abcam, ab52857), or anti-SOX2 (1:100, Abcam, ab171380) primary antibody followed by Alexa Fluro 488 and/or Alexa Fluro594 secondary antibodies (1:200, Life Technologies). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole. Fluorescence images were obtained by using a laser scanning confocal imaging system (Olympus Microsystems).

Immunofluorescence was performed as described previously.^{32 (link), 42 (link)} Briefly, soluble proteins were extracted using extraction buffer to enrich the chromatin-bound nuclear protein signal (20 mM HEPES (pH 8), 20 mM NaCl, 0.1 mM NaF, 0.5% Igepal) for 5 min, the cells were then fixed with 4% paraformaldehyde in PBS for 20 min and cells were left in PBS at 4 °C until stained. For immunostaining, cells were first permeabilised in 0.2% Triton X-100 for 5 min and then blocked (3% BSA in PBS) for 1 h, followed by incubation with primary antibodies as indicated. After incubation, cells were washed three times with PBS before the addition of secondary antibodies as indicated, and these were incubated for 1 h at room temperature (Alexa Fluro 594 with a 1:400 dilution (Life Technologies) in 3% BSA). Nuclear DNA was stained with DAPI (Sigma) for 5 min at 1 μg/ml. A Delta Vision Elite Live Imaging Microscope (Applied Precision, GE HealthCare Lifesciences, Parramatta NSW, Australia) and softWoRx analysis software (Applied Precision) were used for the cell imaging. High-throughput analysis was performed using a Incell 2200 (GE Healthcare Lifesciences, Parramatta NSW, Australia) and a Incell investigator analysis software (GE Healthcare).