(A) Schematic showing generation of the HMGCR-Clover reporter. (i) The endogenous HMGCR locus of HeLa cells was modified by transfection of Cas9, sgRNA and a donor template. The 5’ and 3’ arm of the donor template were designed as homologous sequences encoding the C-terminal region and 3’ UTR of the HMGCR gene. The C-terminal Clover tag (green) was appended in frame to the ORF of HMGCR (blue) including a myc-tag (grey) as spacer and an antibiotic resistance cassette flanked by loxP sites. (ii) Cells having stably integrated the recombination construct were enriched by antibiotic selection. (iii) The resistance cassette was removed by transient transfection of Cre recombinase to yield endogenous, C-terminally modified HMGCR (iv). pUb, ubiquitin promoter; ORF, open reading frame; UTR, untranslated region. (B – E) The HMGCR-Clover reporter phenocopies untagged HMGCR. (B) HMGCR-Clover expression (grey shaded histogram) as detected by flow cytometry under sterol-replete conditions. (C) Immunoblot of HMGCR in sterol-depleted (SD) HeLa WT vs. HMGCR-Clover cells -/+sterols (S) for 2 hr. For sterol depletion, cells were switched to SD medium (10% LPDS, mevastatin (10 μM), mevalonate (50 μM)) for 16 hr. Whole-cell lysates were separated by SDS-PAGE and HMGCR(-Clover) detected with an HMGCR-specific antibody. (D) Cytofluorometric analysis of HMGCR-Clover HeLa cells cultured in sterol-replete (shaded histogram) vs. sterol-depleted medium (SD) (16 hr, blue line histogram). Sterols (S) (2 µg/ml 25-hydroxycholesterol, 20 µg/ml cholesterol) were added back for 2 hr (red line histogram). (E) Flow cytometric analysis of HMGCR-Clover cells treated overnight with Bortezomib (25 nM), mevastatin (10 μM), or NMS-873 (10 μM) for 8 hr. (F) CRISPR/Cas9-mediated depletion of Insig-1 and −2 together (red line histogram) induce a dramatic increase in HMGCR-Clover expression, equivalent to sterol depletion (SD, orange shaded). HMGCR-Clover cells transiently expressing the indicated Insig-1/2 specific sgRNAs (four sgRNAs per gene) were treated as in (D) and, where indicated, sterols (S) added back for 4 hr (SD + S, bottom row). Representative of ≥3 independent experiments. (G) Immunofluorescence analysis of HMGCR-Clover and KDEL (ER marker) expression, showing co-localisation in sterol-depleted (SD, 16 hr) HMGCR-Clover HeLa cells. Scale bar = 20 μm.