Fig 1.
A haploid genetic screen for resistance to T cell mediated killing.
A) Experimental scheme: A library of gene trap-mutagenized haploid HAP1 cells was generated, in which most cells harbor irrelevant mutations (red) and rare cells harbor mutations that reduce their sensitivity towards T cell killing (green). Upon exposure to T cells (depicted in blue), cells with reduced sensitivity are positively selected and, following expansion, integration sites in the surviving cell population are analyzed. B) List of most significantly enriched hits. Yellow: components of the IFNGR signaling pathway; green: SLFN11.
Fig 2.
Interference with the IFNGR pathway and SLFN11 protects HAP1 from T cell- and IFNγ-mediated toxicity.
A) Parental HAP1 or two IFNGR1 KO clones were exposed to T cells at the indicated effector: target ratio for 24 h. 7 days after T cell exposure, surviving cells were stained with crystal violet. B-C) HAP1 cells transduced with a Cas9-encoding lentiviral vector with either a non-targeting sgRNA (sgRNA ctrl) or two independent sgRNA targeting SLFN11 (sgRNA SLFN11#1 and#2) were exposed to either T cells at the indicated effector: target ratio for 24 h (B), or to IFN-γ at the indicated concentrations for the whole duration of the experiment (C). 7 days after T cell or IFN-γ exposure, surviving cells were stained with crystal violet. D) HAP1 cells transduced with a Cas9-encoding lentiviral vector with either a non-targeting sgRNA (sgRNA ctrl) or two independent sgRNA targeting SLFN11 (sgRNA SLFN11#1 and#2) were exposed to the indicated concentrations of IFN-γ. 48 hours after IFN-γ exposure, cell viability was assayed by analysis of metabolic activity. E) HAP1 cells transduced with a control lentiviral vector or with two independent lentiviral vectors encoding SLFN11-targeting shRNA were exposed to the indicated concentrations of IFN-γ. 48 hours after IFN-γ exposure, cell viability was assayed by analysis of metabolic activity. F) Validation of SLFN11 KD by western blot analysis of untreated or IFN-γ treated (10 ng/ml, 24 h) HAP1 cells. G) HAP1 cells that were either untreated or pre-incubated for 1 h with the indicated compounds (20 μM for necrostatin, 10 μM for Q-VD-OPh) were exposed to the indicated concentrations of IFN-γ. 48 hours after IFN-γ exposure, cell viability was assayed by analysis of metabolic activity. * p<0.05, ** p<0.01, *** p<0.001.
Fig 3.
SLFN11 does not regulate IFNGR signaling and evaluation of IFN-γ effects in HAP1.
A) Schematic overview of experimental design in B. B) Growth kinetics of cells left untreated or treated with 10 ng/ml of IFN-γ for 24 h, in the presence or absence of Q-VD-OPh. C). Western blot analysis of phosphorylated STAT1 in HAP1 cells transduced with a control lentiviral vector or with lentiviral vectors encoding independent SLFN11-targeting shRNA, either left untreated or exposed to 10 ng/ml of IFN-γ for 24h. D) IRF1 transcript levels following exposure to 10 ng/ml of IFN-γ for the indicated times in parental, IFNGR1 KO, and SLFN11 KO cells.
Fig 4.
Interference with SLFN11 expression protects HAP1 from IFN-γ and DDA.
A-D) Parental HAP1 cells or three SLFN11 KO clones were exposed to the indicated concentrations of IFN-γ (A), cisplatin (B), doxorubicin (C), or docetaxel (D). 48 hours after IFN-γ or chemotherapy exposure, cell viability was assayed by analysis of metabolic activity. * p<0.05, ** p<0.01, *** p<0.001.
Fig 5.
Sensitization of melanoma and prostate cancer cells to DDA but not IFN-γ.
A-B) Validation of SLFN11 KD by western blot in DU145 (A) and WM2664 (B). C-D) DU145 (C) and WM2664 cells (D) transduced with a control lentiviral vector or with independent SLFN11-targeting shRNA were exposed to the indicated concentrations of IFN-γ. 7 days after IFN-γ exposure, cell viability was assayed by analysis of metabolic activity. E-H) DU145 (E, G) and WM2664 (F, H) cells transduced with a control lentiviral vector or with independent vectors encoding SLFN11-targeting shRNA were exposed to the indicated concentrations of cisplatin (E, F) or doxorubicin (G, H). 48 hours after exposure, cell viability was assayed by analysis of metabolic activity. I-J) DU145 (I) and WM2664 (J) cells that were either untreated or pre-incubated for 1 h with Q-VD-OPh (10μM) were exposed to the indicated concentrations of IFN-γ. 7 days after IFN-γ exposure, cell viability was assayed by analysis of metabolic activity. * p<0.05, ** p<0.01, *** p<0.001.
Fig 6.
Western blot analysis of the indicated proteins in control or SLFN11 KD HAP1 cells following exposure to 10 ng/ml of IFN-γ for the indicated time periods.
Fig 7.
Reversible inhibition of IFN-γ sensitivity by inactivation of the SLFN11 locus.
A) Schematic overview of experimental design. A promoterless gene trap vector was inserted into the first intron of the SLFN11 gene. SLFN11 exons are indicated in red, GFP and puromycin N-acetyltransferase coding sequences in green and orange, and loxP sites in purple. Left part of the panel depicts the genomic locus, right part depicts the resulting transcripts. B-C) Cells with integrated gene trap vector were either left untreated or exposed to the indicated IFN-γ concentrations. Technical replicates (B) and representative flow plots (C) are depicted. D) Puromycin treatment results in homogeneous selection of cells with integrated gene trap vector. E) Detection of the integrated gene trap vector in the SLFN11 locus by PCR. Used primers are depicted in panel A (F and R). 1: untransfected HAP1; 2: transfected and puromycin-selected HAP1; w: water control. F-G) Excision of the gene trap vector results in re-sensitization of HAP1 cells to IFN-γ. Cells from panel D were transfected with a cre expressing vector and either left untreated or exposed to the indicated IFN-γ concentrations. Technical replicates (E) and representative flow plots (F) are depicted. * p<0.05.