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. 2020 Jan 21;219(3):e201908142. doi: 10.1083/jcb.201908142

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© 2020 He et al.

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Figure S1. — Gene editing of SUM159 cells to express CLTA-TagRFP and EGFP-Aux1 or CLTA-TagRFP and EGFP-GAK. (A) CRISPR/Cas9 gene editing strategy used to incorporate EGFP at the N-terminus of Aux1 or GAK. The resulting DNA sequences, including the short linker between the C-terminus of EGFP and N-terminus of Aux1 or GAK, are shown. (B) Genomic PCR analysis showing biallelic integration first of EGFP into the DNAJC6 (Aux1) genomic locus to generate the clonal gene-edited cell line EGFP-Aux1^+/+ (left) and then of TagRFP into the CLTA genomic locus of the same cells to generate the clonal double-edited cell line EGFP-Aux1^+/+ CTLA-TagRFP^+/+ (center). Right: Western blot analysis of cell lysates from the EGFP-Aux1^+/+ cells probed with antibodies for Aux1/GAK and actin. Although the genomic PCR shows biallelic integration of EGFP sequence into the Aux1 genomic locus, the Western blot indicates expression of a small amount (∼15%) of untagged Aux1. The expression of EGFP-Aux1 in EGFP-Aux1^+/+ cells was higher than endogenous Aux1 in the parental SUM159 cells; this up-regulation of EGFP-Aux1 expression is due to either single-cell cloning selection or the genome editing. (C) Genomic PCR analysis showing biallelic integration first of TagRFP into the CLTA genomic locus to generate the clonal gene-edited cell line CLTA-TagRFP^+/+ (left) and then of EGFP into the GAK genomic locus of the same cells to generate the clonal double-edited cell line EGFP-GAK^+/+ and CTLA-TagRFP^+/+ (center). Right: Western blot analysis of cell lysates from the EGFP-GAK^+/+ and CTLA-TagRFP^+/+ cells probed with antibodies for GAK and actin. (D) Effect of expression of EGFP-Aux1 and CTLA-TagRFP on receptor-mediated uptake of transferrin. The histogram shows similar amounts of internalized Alexa Fluor 647–conjugated transferrin in parental and gene-edited EGFP-Aux1^+/+ and CTLA-TagRFP^+/+ cells probed by flow cytometry (n = 5 experiments, mean ± SD, P value by two-tailed t test). (E) Effect of expression of EGFP-GAK and CTLA-TagRFP on receptor-mediated uptake of transferrin (n = 5 experiments, mean ± SD, P value by two-tailed t test). (F) Scatter plots comparing maximum fluorescence intensities of EGFP-Aux1 and CLTA-TagRFP with each other (left; Pearson correlation coefficient r = 0.331) and maximum fluorescence intensity of EGFP-Aux1 with the lifetime of the endocytic coated structure in which it was found (right; Pearson correlation coefficient r = 0.115), from 938 traces in eight cells. Data from bottom surfaces of double-edited EGFP-Aux1^+/+ and CLTA-TagRFP^+/+ cells imaged at 1-s intervals for 300 s by TIRF microscopy. (G) Scatter plots comparing maximum (max.) fluorescence intensities of EGFP-GAK and CLTA-TagRFP with each other (left; Pearson correlation coefficient r = 0.373) and max. fluorescence intensity of EGFP-GAK with the lifetime of the endocytic coated structure in which it was found (right; Pearson correlation coefficient r = 0.153), from 900 traces in eight cells. Data from bottom surfaces of double-edited EGFP-GAK^+/+ and CLTA-TagRFP^+/+ cells imaged at 1-s intervals for 300 s by TIRF microscopy.