Fig. 3: MLH1 regulates protein trafficking of HER2.

A Gene set enrichment analysis of RNAseq data comparing isogenic shLuc and shMLH1 MCF7 cells after treatment with fulvestrant for 4 days. P values were generated using DESeq2 R package and adjusted for multiple comparison using Benjamini–Hochberg. Comparable RPPA data analysis in Fig. S4A. Raw read counts available as supplementary data. B Co-immunofluorescence for HER2 and lysosomal marker, LAMP1 (orange arrows) at baseline and after 18, 36, and 54 h of fulvestrant treatment. Green arrows indicate HER2 that is not colocalized with LAMP1, and red arrows indicate LAMP1 positivity alone. Validation in T47D cells in Fig. S4B. C Immunofluorescence staining for HER2 in MCF7 shLuc and shMLH1 cells treated with vehicle and 36 h of fulvestrant alone or a combination of fulvestrant and chloroquine, an autophagy inhibitor. For shLuc vs. shMLH1 vehicle, p = 0.0006. Validation in T47D cells in Fig. S4C. Quantification (D) and representative photomicrographs (E) from 36 h of live cell tracking of colocalization of HER2 and LC3 (orange arrows), a marker of autophagosomes, in MCF7 shLuc and shMLH1 cells treated with vehicle or fulvestrant. Green arrows indicate HER2⁺ LC3⁻ cells. Cells were tracked after administration of fulvestrant. All quantification is of three independent biological replicates conducted through ImageJ and is represented as strip charts. Two-sided Student’s t test determined all p values. For shLuc vehicle vs. 36-h fulvestrant treatment, p = 0.0003 and for shLuc vs. shMLH1 at 36-h fulvestrant treatment, p = 0.0005. Scale bars represent 50 µ. Source data for all figures available with paper.