Ab18447

As previously described [52 (link)], cells were suspended in PBS at a concentration of 1.2×10⁷ cells/ml and incubated with mouse anti-TF antibody (ADG4508, Sekisui, MA) 1:25 or isotype mouse IgG antibody (AB18447, Abcam, SF) 1:50 for 20 minutes at RT. After PBS washes, cells were incubated for 20 minutes with APC-linked goat anti-mouse-IgG antibody (A865, Invitrogen) at RT before resuspending in PBS. The Aldefluor ^TM assay kit (Stem Cell Technologies, Vancouver, Canada) was used to measure ALDH1 enzymatic activity according to manufacturer's instructions. As previously described [5 (link)], cells were resuspended at a concentration of 1×10⁶ cells/ml. 5μl of Aldefluor reagent was added and 500 μl was immediately transferred to a tube containing the ALDH1 inhibitor DEAB. Samples were incubated at 37°C for 30 minutes before centrifugation and collection of cell pellet. Baseline fluorescence was established by inhibiting ALDH1 activity with DEAB and used to generate a gate (top 0.1%) to identify ALDH1-high cells that have not been incubated with DEAB. Fluorescence was measured using FACSCalibur (BD Biosciences) and analysed using FlowJo Version 10.1.1.

Sulfo-N-succinimidyl oleate (SSO, sc-208408), bafilomycin A1 (sc-201550) and siRNAs for CD36 (sc-29995), SR-BI (sc-44752 and sc-44753) and negative control (siRNA-A, sc-37007) were from Santa Cruz Biotechnology, Inc. The compounds W-9 and AP5055 were synthesized in the Medicinal Chemistry Laboratory of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, in a purity greater than 98.5%. The structures of the compounds were confirmed with proton nuclear magnetic resonance spectroscopy and mass spectrometry spectra. Telaprevir/VX-950 (HY-10235) was from MedChemExpress, Inc. The antibody to SR-BI (NB400-104) was from Novus Biological, Inc. The mAbs to human CD36 (ab17044, ab23680, ab76521 and ab133625), HCV core (ab2740), HCV NS3 (ab13830), control IgG₁ (ab18447) and Nrf2 antibody (ab31163) were from Abcam, Co. Ltd. The mAb to beta-Actin (TA-09) was from Beijing ZSJQ-BIO, Co. Ltd. The antibodies to HCV E1 (GTX103352) and E2 (GTX103353) were from Gene Tex, Inc. The mAb to HA tag (6E2) (2367S) and the secondary antibodies (7074S and 7076S) were from Cell Signaling Technology, Inc.

To examine the role of HMGB1 and RAGE in 8-nitroG formation, A549 cells were pretreated with 10 μg/ml anti-HMGB1 (ab77302, Abcam Cambridge, UK) and 10 μg/ml anti-RAGE (ab54741, Abcam, Cambridge, UK) antibodies. We also used the corresponding isotype control IgGs [mouse IgG₁ (ab18447, Abcam) for anti-HMGB1 antibody and IgG_2a (ab18414, Abcam) for anti-RAGE antibody] to confirm the specificity of these antibodies. Then the cells were incubated with 200 ng/ml of In₂O₃, ITO and InCl₃ for 4 h. Then 8-nitroG formation was examined by immunocytochemistry as described above.