Fig 1.
A. Cell viability after delivery of HNRNPK or non-targeting (NT) qgRNAs (CellTiter-Glo assay).
Results are normalized against the NT condition. 10 individually treated wells. B-D. Cas9 and hnRNP K protein upon delivery of HNRNPK (+) or NT (-) qgRNA. E. Genome-wide CRISPR deletion screen. F. Volcano plot showing differential sgRNAs abundance in the HNRNPK vs. NT comparison at day 14. G-H. Cell viability after sequential co-deletion of each candidate gene and HNRNPK (CellTiter-Glo assay). Results are normalized against the seeded cell density before HNRNPK ablation and compared to the double NT+NT condition. Red empty circles: non-targeting control (NT) and non-specific genes (PCNA, GPKOW, PRNP). Black-filled circles: genes confirmed only in LN-229 C3 cells. Yellow-filled circles: genes confirmed in both LN-229 C3 and U251-Cas9 cells. n ≥ 3. Data information: n represents independent experiments. Mean ± SEM. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001 (Two-way ANOVA Tukey’s test in A. One-way ANOVA Dunnett’s test in G-H).
Fig 2.
A-B. Clonogenic fitness of ΔTFAP2C or NT-unmodified cells after delivery of HNRNPK or NT qgRNAs (Crystal violet staining).
Western blot: hnRNP K and Tfap2c protein 7 (A) and 10 (B) days after qgRNAs delivery. n = 3. C, E. Tfap2c protein after HNRNPK ablation. n = 5. D, F. qRT-PCR showing TFAP2C RNA in LN-229 C3 (D) and U251-Cas9 (F) cells upon HNRNPK deletion. n = 3 and 5. G. Viability course of LN-229 C3 and U251-Cas9 cells untransduced or overexpressing TFAP2C or mCherry after delivery of HNRNPK (+) or NT (-) qgRNAs (CellTiter-Glo assay). Results are normalized against the NT condition. 10 individually treated wells. Western blot: hnRNP K, Tfap2c, and mCherry protein 7 (LN-229 C3) or 10 days (U251-Cas9) after qgRNAs delivery. H-I. Co-immunoprecipitation of Tfap2c and hnRNP K in low-detergent conditions after benzonase nuclease digestion. IP: Immunoprecipitated Protein; FT: Flow-Through after immunoprecipitation. Data information: qRT-PCR results are normalized against GAPDH expression. n represents independent experiments. f.c.: fold change. Mean ± SEM. *: p < 0.05, **: p < 0.01, ****: p < 0.0001 (Unpaired t-test in D and F. Two-way ANOVA Šídák’s test in G).
Fig 3.
A-B. PARP and Caspase 3 protein cleavage ratio after HNRNPK and TFAP2C deletion.
4 hours 1 μM staurosoprine (STS) was used as positive control. n = 3. C-D. Viability of cells 7 (C) or 10 (D) days after HNRNPK or NT qgRNAs transduction and supplementation of Z-VAD-FMK (CellTiter-Glo assay). Results are normalized against the DMSO/NT condition. Multiple comparison was made between Z-VAD-FMK and DMSO treatments in ΔHNRNPK cells. 6 individually treated wells. E-F. Viability of ΔTFAP2C cells treated with staurosporine or DMSO (CellTiter-Glo assay). Results are normalized against the DMSO-treated cells. 4 individually treated wells. Data information: n represents independent experiments. Mean ± SEM. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 (Two-way ANOVA Uncorrected Fisher’s LSD in A-B, Dunnett’s test in C-D, and Šídák’s test in E-F).
Fig 4.
A-B. RNA-seq Gene Set Enrichment Analysis. Shown are the 20 most negatively (A) and positively (B) enriched GOBP terms (Gene Ontology Biological Process), respectively, in ΔHNRNPK vs. NT (A) and ΔTFAP2C;ΔHNRNPK vs. ΔHNRNPK (B). NES: Normalized Enrichment Score. C. Intersection of RNA-seq data resulting from the ΔTFAP2C;ΔHNRNPK vs. ΔHNRNPK and the ΔHNRNPK vs. NT comparisons. D. Gene enrichment of the biological process for the inversely regulated genes in C.
Fig 5.
A. Intracellular ATP level in ΔTFAP2C or NT-unmodified LN-229 C3 cells 4 days upon delivering HNRNPK and NT qgRNAs. 5 individually treated wells. B. Representative blot for LC3-II protein in LN-229 C3 cells after HNRNPK and TFAP2C ablation. Quantification includes blot shown in S6C Fig. 4 hours 100 μM chloroquine (CQ). n = 4. C-D. mTOR RNA (C) and protein (D) upon deletion of HNRNPK and TFAP2C in LN-229 C3 cells. 6h HBSS-starvation (Starv.) in D was used as a positive control. qRT-PCR: n = 6. WB: n = 3. E. 4EBP1 and S6 protein phosphorylation after HNRNPK and TFAP2C ablation in LN-229 C3 cells. 6h HBSS-starvation (Starv.) was used as positive control. n = 3. F. mTOR and 4EBP1 and S6 protein phosphorylation in LN-229 dCas9-VPR cells upon TFAP2C overexpression. n = 4. G. Hypothesized mechanism of TFAP2C and HNRNPK in cell metabolism regulation. Data information: qRT-PCR results are normalized against GAPDH expression. n represents independent experiments. f.c.: fold change. Mean ± SEM. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 (Two-way ANOVA Uncorrected Fisher’s LSD in A-E. Unpaired t-test in F).
Fig 6.
A. Proteinase K (PK) digested (bottom) and undigested (top) western blots showing, respectively, PrPSc and total PrP after HNRNPK silencing (96 hours) and TFAP2C overexpression (192 hours) in PG127-infected HovL cells. n = 3. B. Quantification of PrPSc from A and S8A Fig. n = 6. C. PrPC protein in uninfected HovL cells upon HNRNPK knockdown (96 hours) and TFP2C overexpression (192 hours). n = 3. D. Flow cytometry quantification of anti-PrPSc 6D11 antibody signal in PG127-infected HovL cells upon HNRNPK knockdown and TFP2C overexpression at different time points. 3 individually treated wells. Data information: PG127-infected (PG127) and Not-infectious Brain Homogenate (NBH) were used for PK digestion positive and negative control, respectively. Non-targeting siRNA (siNT) and mCherry overexpression were used as controls. n represents independent experiments. f.c.: fold change. Mean ± SEM. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 (Two-way ANOVA Uncorrected Fisher’s LSD in B-D).
Fig 7.
A. qRT-PCR showing HNRNPK, MTOR, RPTOR, and RICTOR RNA upon HNRNPK silencing (96 hours) in PG127-infected HovL cells. n = 6. B. 4EBP1 and S6 protein phosphorylation upon HNRNPK silencing (96 hours) or treatment with 500 nM Torin-1 or Rapamycin (72 hours) in PG127-infected HovL cells. n = 2. C. PK digested (bottom) and undigested (top) western blots showing, respectively, PrPSc and total PrP after treatment with 500 nM Torin-1 or Rapamycin (72 hours) in PG127-infected HovL cells. n = 4. D. Flow cytometry quantification of anti-PrPSc 6D11 antibody signal in PG127-infected HovL cells upon HNRNPK knockdown (96 hours) and treatment with 500 nM Torin-1 or Rapamycin (72 hours). n = 3, each with 3 individually treated wells. Data information: PG127-infected (PG127) and Not-infectious Brain Homogenate (NBH) were used for PK digestion positive and negative control, respectively. Non-targeting siRNA (siNT) and DMSO were used as controls. qRT-PCR results are normalized against GAPDH expression. n represents independent experiments. f.c.: fold change. Mean ± SEM. ns: p > 0.05, *: p < 0.05, ***: p < 0.001, ****: p < 0.0001 (Multiple Unpaired t-test Holm- Šídák method in A. One-way ANOVA Dunnett’s test in C. Two-way ANOVA Šídák’s test in D).