Abcg2 ab3380

Sections from paraffin embedded kidney biopsies with chronic kidney disease (normal and reduced eGFR) and kidney tissue obtained from tumor nephrectomy specimens (n = 54) were stained with antibodies against HIF1α, VEGFA, and ABCG2. After antigen retrieval, slides were incubated with the following antibodies: HIF1α clone mgc3 ab16066 (Abcam, Cambridge, UK), dilution 1:400; VEGFA sc-152 (Santa Cruz Biotechnology), dilution 1:300; ABCG2 ab3380 (Abcam, Cambridge, UK), dilution 1:60. The immunohistochemical staining was conducted on automated staining systems (ABCG2 on Ventana BenchMark, Ventana Medical Systems, USA; HIF1α and VEGFA on Leica Bond System, Leica Biosystems) following the manufacturer’s instructions. Visualization was performed using the avidin–biotin complex method leading to a brown staining signal. For all stainings the intensity in the glomerular and tubulointerstitial compartment (negative = score 0, weak = score 1, strong = score 2) in comparison to expression in normal tissue was assessed. Statistical analysis using Fisher’s exact test was performed with SPSS statistics software version 22 (IBM, USA). p-values < 0.05 were considered as significant.

The E-cadherin–GFP expression construct was a gift from Alpha Yap^{50 (link)} (University of Queensland, Queensland, Australia). Wild-type E-cadherin was generated by PCR amplification of the corresponding cDNA fragments using E-cadherin–GFP as a template. The E-cadherin tyrosine-to-alanine (⁷⁵³YYY^755→753AAA⁷⁵⁵) mutant was generated by site-directed mutagenesis using wild-type E-cadherin as a template. The correct clone sequence was verified by sequencing. Lentiviral-TOP-dGFP-reporter plasmids were obtained from Addgene (Addgene plasmid 14715, Cambridge, MA, USA). A pLKO.1-shRNA encoding an shRNA with a scrambled sequence or sequences targeting human Sox15 and E-cadherin, purchased from the National RNAi Core Facility, Taiwan, was introduced into HEK293T cells using the lentiviral packaging vectors pMD.G and pCMV_8.91. Antibodies against the following proteins were used: E-cadherin (610181; BD Biosciences, San Jose, CA, USA); histone H3 (Ab1791) and ABCG2 (Ab3380) (all from Abcam, Cambridge, MA, USA); active β-catenin (ALX-804-260/1; dephosphorylated at Ser33/37; Enzo Life Sciences, Farmingdale, NY, USA); total β-catenin (C2206), β-actin (A5441) and TCF4 (T5817) (all from Sigma-Aldrich, St Louis, MO, USA); CD133 (3663) and phosphorylated β-catenin (phosphorylated at Ser33/37/Thr41) (9561) (all from Cell Signaling Technology, Beverly, MA, USA).