Phenix imager

HT1080 CRISPRi cells were transduced with sgRNA-expressing lentiviral vectors and selected with antibiotics. 9 days after sgRNA transduction and 1 day after final re-plating, cells were assayed for their lysosomal hydrolase activity by incubating with 0.2 ug/mL Hoechst 33342 and either 200 uM PFB-FDGlu (for glucosylceramidase; P11947, Invitrogen) or 33 uM C₁₂FDG (for beta galactosidase; I2904, Invitrogen) in Imaging Media for 1 hour at 37°C, before imaging on the Phenix imager (Perkin-Elmer) with a 63x objective in confocal mode.
Image analysis was performed using the Harmony software (Perkin-Elmer), where flat-field corrected images were segmented for nucleus and cytoplasm. Total fluorescence intensity for each cell was extracted, and each biological replicate (two per condition) was represented by the median of the per-cell fluorescence (MFI) from all segmented cells, relative to the Non-targeting controls, as log10 fold-change.

HT1080 cells were transduced with lentiviral vectors expressing either TagBFP-tagged GALNT2 (Golgi) or mRFP1-tagged TMEM192 (Lysosome), and selected with antibiotics. Cells with stable integration were fixed with 4% formaldehyde (15 minutes at 4°C), permeabilized with 20 ug/mL digitonin (30 minutes at room temperature), blocked with 1% BSA (30 minutes at room temperature), and incubated with primary antibody against TMEM251 (HPA048559, Sigma-Aldrich; 1:200 overnight at 4°C) followed by Alexa Fluor 488conjugated secondary antibody (1:1000, 2 hours at room temperature). Samples were imaged on the Phenix imager (Perkin-Elmer) with a 63x objective in confocal mode.

HT1080 CRISPRi cells were transduced with sgRNA-expressing lentiviral vectors and selected with antibiotics. For dual-target samples, cells were transduced simultaneously with two vectors and co-selected with two antibiotics. 8 days after sgRNA transduction and 2 days after final re-plating, cells were fixed, permeabilized, blocked, and stained as above, using primary antibody against LAMP1 (ab25630, Abcam; 1:50) and Alexa Fluor 647-conjugated secondary antibody. Alexa Fluor 555-conjugated WGA at 1.5 ug/mL and Hoechst 33342 at 5 ug/mL were included during secondary antibody incubation. Samples were imaged on the Phenix imager (Perkin-Elmer) with a 63x objective in confocal mode.
Image analysis was performed using the Harmony software (Perkin-Elmer), where images were flat-field corrected and regions corresponding to the nucleus, cytoplasm, and lysosome were identified. WGA signals that colocalized with the lysosomes were quantified by the median fluorescence intensity (MFI) for each cell. Each biological replicate (two per condition) was represented by the upper quartile of the per-cell MFIs from all segmented cells.