Cell culture methods

Deletion of XBP1 via CRISPR/Cas9

hTert-RPE1 cells containing an XBP1 deletion were generated using CRISPR/Cas9. The Benchling software (www.benchling.com) was used for the design of the guide RNAs (gRNAs). gRNA1 recognizes a region directly upstream of the XBP1 start codon, whereas gRNA3 is complementary to a region within exon 5. 70-80 % confluent RPE1 cells were transfected with pX458-derived plasmids containing both Cas9-2A-GFP and the gRNAs using Fugene 6 or the Nucleofection technology. pX458 (pSpCas9(BB)-2A-GFP) was a gift from Feng Zhang (Addgene plasmid #48138). 48 h post-transfection, GFP-positive cells were sorted with a Sony SH800 cell sorter into 96-well plates containing conditioned DMEM-F12-medium. After sorting, every well contained a single GFP-positive cell. Cells were incubated for several days until colonies were formed. Afterwards, gDNA was extracted and analyzed for an XBP1 deletion via PCR. The following combinations of oligonucleotides were used: KS548+KS550,

132 Material and Methods

KS550+KS551, KS549+KS551, KS548+KS551. Clones lacking XBP1 were further analyzed by Western blot using an XBP1^s-specific antibody.

Generation of stable cell lines via a lentiviral system

All generated stable cell lines were lentivirus-generated pools based on RPE1 XBP1^-/-. The cell lines were generated using a third-generation-lentivirus system. The genes to be stably introduced into the genome were subcloned into Gateway ENTRY plasmids using either conventional restriction-enzyme based cloning or Gibson assembly cloning. Based on these vectors, lentiviral transfer vectors were created through Gateway LR recombination into lentiviral destination vectors derived from pCDH-PGK-MCS-IRES-PURO (System Biosciences). VSV-G pseudotyped lentiviral particles were packaged using the system described in Dull et al., 1998. The required plasmids pRSV-Rev (Rev), pMDLg/pRRE (Gag/Pol) and pMD2.G (VSV-G) were gifts from Didier Trono (Addgene plasmids #12251, #12253, #12259).

Briefly, Lenti-X cells were transfected with the plasmid containing the construct to be introduced, Gag/Pol, Rev and VSV-G using Lipofectamine 2000. Three days post-transfection lentiviral particles were harvested from the Lenti-X cell supernatant. RPE1 XBP1^-/- cells were transduced with three different virus titers.

Antibiotic selection which resulted in cell populations with stable expression was started two days after transduction and lasted at least for 8 days.

Production of lentiviral particles

Construct to be introduced Gag/Pol

70-80 %confluent RPE1 cells and its derivatives were seeded in 6 cm dishes (100-250 cells per dish). Cells were either incubated overnight or until they were

attached before TM treatment. TM (0.5-0.6 µg/mL) was added to the cells for 4.5 h. Afterwards, the cells were washed twice in DMEM-F12-medium without TM before further incubation (8-10 days). After the corresponding incubation time, the medium was removed and the attached cells washed with PBS. Next, the cells were fixed with 2 mL 70 % ethanol for 15 min at RT. The ethanol was removed and the dishes rinsed in H²O before being put upside down on paper towels for drying. After at least 60 min of drying, the colonies were stained with 0.01 % crystal violet (in H²O) and washed again with H²O. For the evaluation, only colonies containing more than 50 cells and only assays in which the number of colonies in KO was at least 35 % reduced compared to WT, were considered.

Dual luciferase reporter assay

RPE1 cells (70-80 % confluency) were seeded into 6-well plates (1.5 x 10⁵ cells per dish). Cells were transfected using Fugene 6 with 1 µg plasmid containing the UPRE-luciferase reporter. This plasmid (p5 x ATF6-GL3, Addgene plasmid

#11976) was a gift from Ron Prywes. 24 h post-transfection, the cells were washed twice with PBS and passively lysed with 500 µL PLB buffer for 15 min on a rocking platform at RT. 20 µL of the cell lysates was transferred to a 96-well plate. All following steps were performed according to the manufacturer’s protocol (Dual-Luciferase Reporter Assay System for the products E1910 and E1960).

The firefly and renilla luciferase activity was measured on a BioTek Gen5 wellplate reader. The activity of the renilla luciferase was used for normalization purposes.

Flow cytometry

For flow cytometry analyses, 70-80 % confluent RPE1 cells were seeded into 10 cm dishes (10⁶ cells per dish). If needed, cells were treated with TM for 4.5 h before fixation. 24 h after seeding, cells were trypsinized and collected in 15 mL falcons (5 min, 1000 rpm). Cells were washed with PBS and fixed with 70 % ethanol (ice-cold). The ethanol was added drop-wise while vortexing to prevent formation of cell clumps. Cells were afterwards stored at -20°C for at least 30 min.

For staining, the cells were collected into 1.5 mL tubes (5 min, 1000 rpm) and washed in PBS. Afterwards, the cell pellet was resuspended in 50 µL flow

134 Material and Methods

detergent buffer for blocking of non-specific binding. The cells were incubated for 5 min in the detergent buffer before adding 50 µL of α-H³P antibody diluted in flow detergent buffer (final concentration 1:500) to the cells. The cells were incubated for 1 h with the antibody at RT. Then, the cells were washed with PBS and incubated for 30 min at RT with flow detergent buffer containing the secondary Alexa488-goat α-rabbit IgG antibody (1:500). After the incubation, the cells were washed again with PBS and resuspended in 500 µL PBS containing Hoechst 33342 (1.5 µL in 1 mL PBS) and filtered into a flow test tube through a cell strainer snap cap. Finally, the stained cells were incubated in darkness overnight at 4°C before running the flow cytometry analyses. Measurements were performed in collaboration with Viola Nähse (Oslo University Hospital, Institute for Cancer Research). Sample preparation for cell cycle analyses in U. maydis was performed according to Boye et al., 2016. Measurements were performed in collaboration with Beata Grallert (Oslo University Hospital, Institute for Cancer Research).

Analyses were performed either on a BD LSR II UV laser flow cytometer equipped with UV, 405, 488 and 633 nm line lasers or on a BD LSR II Yellow laser flow cytometer equipped with 406, 488, 561 and 640 nm line lasers. Data was analyzed using the BD FACSDiva software.

Flow cytometry analyses were supported by the Flow Cytometry Core Facility (FCCF) of the Oslo University Hospital.

Flow detergent buffer