Journal: Molecules and Cells

Article Title: Expression of HYOU1 via Reciprocal Crosstalk between NSCLC Cells and HUVECs Control Cancer Progression and Chemoresistance in Tumor Spheroids

doi: 10.14348/molcells.2020.0212

Figure Lengend Snippet: (A) Bright-field images of 3D spheroids co-cultured with HUVECs and NSCLC cells (NCI-H460 or A549) and spheroids cultured with lung cancer cells alone. The images were obtained using the Operetta ® High Content Screening System with a 10× objective. (B) Expression levels of ATF6, HYOU1, IRE1, and pAKT (Ser473) in spheroids co-cultured with HUVECs and NSCLC cells (NCI-H460 or A549) and spheroids cultured with lung cancer cells alone, as assessed by western blot analysis. (C) Multilayer image showing immunofluorescence staining of HYOU1 in NSCLC (NCI-H460 or A549) spheroids co-cultured with HUVECs and Hoechst staining of both cell types. (D) Expression levels of cleaved caspase-3 and HYOU1 in lung cancer cells transfected with nonspecific siRNA (siCont) or HYOU1 siRNA (siHYOU1) and co-cultured with HUVECs with or without 10 μM or 20 μM gefitinib or cisplatin for 72 h, as assessed by western blot analysis.

Article Snippet: After washing for three times in 10 min with TBST buffer, the NC membranes were incubated with mouse monoclonal anti-CD133 (W6B3C1; Miltenyi Biotec, Germany), rabbit polyclonal anti-Collagen I (Novus Biologicals, USA), mouse monoclonal anti-p53 (DO-2; Santa Cruz, USA), mouse monoclonal anti-MetRS/MARS (MARSD10B4), rabbit monoclonal anti-CARS (EPR7121), mouse monoclonal anti-HIF-1 alpha (mgc3), rabbit polyclonal anti-N Cadherin, mouse monoclonal anti-Vimentin (RV202), rabbit monoclonal anti-alpha smooth muscle Actin (E184) (all from Abcam, UK), rabbit polyclonal anti-HYOU1, rabbit monoclonal anti-phospho-Akt (Ser473) (D9E), rabbit monoclonal anti-PERK (D11A8), rabbit monoclonal anti-ATF-6 (D4Z8V), rabbit monoclonal anti-IRE1α (14C10), rabbit monoclonal anti-cleaved Caspase-3 (Asp175) (5A1E), rabbit monoclonal anti-cleaved PARP (Asp214) (D64E10), rabbit polyclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (all from Cell Signaling Technology), mouse monoclonal anti-interferon-alpha, goat polyclonal anti-interferon-beta, goat polyclonal anti-interferon-gamma (all from R&D Systems, USA), and mouse monoclonal anti-β-actin (Sigma-Aldrich) for 16 h at 4°C.

Techniques: Cell Culture, High Content Screening, Expressing, Western Blot, Immunofluorescence, Staining, Transfection