Alexa594 secondary antibody

96-well seeded DU145 and 22Rv1 cells were treated with 10 µl of Cell Counting Kit-8 solution (CCK-8) (Dojindo, MD, USA) for 1 h before conducting the measurements. The plates were read at an optical density of 450 nm using a SpectraMaxiD3 spectrophotometer (Molecular Devices, CA, USA).
Proliferating cell nuclear antigen (PCNA) in DU145 and 22Rv1 cells were stained with primary antibody against PCNA (ab18197; Abcam, Cambridge, UK) followed by Alexa594 secondary antibody (A11037; Thermo Fisher) staining. DAPI-containing Vectashield (Vector Laboratories, CA, USA) were used for mounting and microscopic imaging (CKX53, Olympus, Tokyo, Japan) with U-HGLHPS illumination system (Olympus) and eXcope X7M camera (DIXI Science, Daejeon, Korea).

Caco-2/BBe cells were cultured on glass coverclips for 1 day or 21 days and fixed with 4% PFA and immuno-stained with Alexa 647 direct-labeled HES1 antibody (1:200; ab196577, Abcam, Cambridge, UK), NR3C1 antibody (1:400; #3660, Cell Signaling Technology, Danvers, MA, US) and Alexa 594 secondary antibody (1:600; Thermo Fisher, Waltham, MA, USA). Nuclei were labeled with DAPI (4,6-diamidino-2-phenylindole). 1024 × 1024, 16-bit 3D confocal images were acquired using a Zeiss LSM 710 laser scanning confocal microscope with a 63x Plan-Apochromat 1.4na DIC objective. For HES1 quantification, images were taken keeping the same laser settings for HES1 channel for all samples. Three 1024 × 1024 Day21 3D images and seven 1024 × 1024 Day1 3D images were collected. Maximum intensity projections of 3D image stacks were analyzed to compare average intensities of HES1 in Day1 and Day 21 Caco-2/BBe cells using Fiji^{32 (link)}. 8-bit images were created and the DAPI channel was used to create nuclei masks. Overlapping nuclei and nuclei on the edge of the image were then manually eliminated (Figure S5). These masks were redirected to the corresponding HES1 and NR3C1 channels to analyze mean intensities of HES1 and NR3C1 within the nucleus. T- test was performed on the mean intensities per nuclei of 100 total nuclei for each sample set (Day1 & Day21).