Alexa fluor 647 conjugated anti rat igg

4T1 cells (murine metastatic breast cancer cell line), tumor-derived endothelial cells and mammary fibroblasts in the ratio of 1:3:2, respectively, were co-cultured in ultra-low attachment U bottom 96-well plates up to 48 h to develop heterogenous CTCs. The clusters were collected using 100 μL of micropipette for further assays. Presence of CTC specific markers such as E-cadherin, CD44 and CK19 were confirmed in the heterogenous CTCs through immunostaining technique. Rat anti-CD44 mAb and rabbit anti E-cadherin (Novus Biologicals, USA) antibody tagged with Alexafluor-647 conjugated anti-rat IgG (Cell Signaling Technology, USA) and Alexafluor-488 anti-rabbit IgG antibody (Molecular Probes, India). Images were taken using laser scanning confocal microscopy (FV1000, Olympus, USA). In the subsequent experiments, clusters were transferred to fresh plates and dosed with nanotheranostic particle suspension (Dox equivalent in 25 μg/mL) for 24 h before analysis. For hyperthermia treated groups, clusters were transferred to eppendorfs and subjected to hyperthermia at 480 kHz for 5 min and stained with specific dyes and antibodies for assays.

Bruceine D was isolated from the fruits of Brucea javanica (Linn.) Merr in our laboratory¹⁶ . For in vitro experiment, BD was dissolved in 100% DMSO (Sigma, 01934-1L) at a final stock concentration of 50 mmol/L and stored at −20 °C as single used aliquot. The antibodies for GAPDH (5174s, 1:1000), β-actin (3700s, 1:1000), HIF-1α (36169s, 1:1000), β-catenin (8480s, 1:1000), PKM2 (4053T, 1:1000), HK2 (2867T, 1:1000), LDHA (3582T, 1:1000), Cyt c (11940T, 1:1000), HRP-conjugated anti-rabbit IgG (1:5000), HRP-conjugated anti-mouse IgG (1:5000), and Alexa Fluor 647 conjugated-anti-rat IgG (4418s, 1:1000) were purchased from Cell signaling technology (CST, Danvers, MA, USA). Primary antibodies for GLUT1 (ab115730, 1:1000), GLUT3 (ab191071,1:1000), HIF-1β (ab239366, 1:1000), ICAT (ab129011, 1:1000), HIF-1α (ab51608, 1:1000), and recombinant ICAT (ab101468) were both obtained from Abcam (Cambridge, UK). MCT4 (sc-376140, 1:1000) mouse IgG (sc-2025, 1:50) was procured from Santa Cruz (OR, USA). Recombinant human ICAT protein (ab101468) was obtained from Abcam (Cambridge, UK). Cycloheximide (Sigma, 5087390001) and MG132 (Sigma, SML1135) was obtained from Sigma (Shanghai, China).