Anti cd44

To measure the ALDH1 activity, Aldefluor assay was performed according to manufacturer’s (Stemcell Technologies, Durham, NC, USA) instruction. Dissociated single cells were suspended in Aldefluor assay buffer containing ALDH substrate, and ALDH inhibitor, diethylaminobenzaldehyde (DEAB), was used as negative control.
Cells were stained with anti-CD44 (Miltenyi Biotech., Auburn, CA, USA), or anti-^memGRP78 (Cell Signaling, Danvers, MA, USA) or anti-ABCG2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies conjugated to phycoerythrin according to the manufacturer’s instructions. Red (>650 nm) fluorescence emission from 10,000 cells illuminated with blue (488 nm) excitation light was measured with FACSCalibur (Becton Dickinson, Mountain View, CA, USA) using CellQuest software [44 (link), 57 (link)].

HeLa, HCT116, and 22Rʋ1 cells were grown in an ultra-low attachment plate for six days with and without treatment of GSK J4. Cells were washed two times in ice-cold PBS and resuspended in 100 µL of staining solution (5 µL PE-conjugated anti-CD 70 (BD Bioscience), CD 133 (Miltenyibiotec-130-090-853), and FITC-conjugated anti-CD 105(BD Bioscience- 56143) and anti-CD44(Miltenyibiotec -130-098-210), 1% FBS, in 1X PBS and incubated at room temperature under the dark condition for 2 h. Then, cells were washed three times in a wash buffer (1%FBS in 1X PBS) and then analyzed by using a Guava Easy-Cyte flow cytometer. For all assays, 10,000 cells were taken for measurement and also for analysis. For each plot, 1000 cells were displayed.