Journal: bioRxiv

Article Title: Analysis of patient data reveals novel cancer-relevant functions for GCN2/eIF2αK4

doi: 10.1101/2025.03.18.643965

Figure Lengend Snippet: A : GSEA of GCN2-correlating genes. Enriched gene sets (FDR < 0.05) from the MSigDB Hallmark gene set for negatively (left) and positively (right) correlating genes. Selected gene sets indicating possible new functions of GCN2 pursued in this study are shown in bold. The results of the GSEA of the top-ranked 1000 correlating genes in each direction are shown in Table S3. B, C Representative immunoblots to show GCN2 levels in ( B ) HeLa and ( C ) hTert-RPE1 cell lines. Cells were transfected with GCN2-targeting siRNA-s to deplete (siGCN2) or were engineered to stably overexpress GCN2 by lentiviral transduction (GCN2 high ). To estimate the efficiency of depletion, different amounts (100%, 10% and 25%; corresponds to 30, 3 and 7.5 µg protein, respectively) of the lysate from cells transfected with control siRNA was loaded, along with 30 µg each of the transfected and the overexpressing samples. γ-tubulin is shown as a loading control. D, E mRNA levels of selected transcripts in ( D ) HeLa and ( E ) hTert-RPE1 cells. mRNA levels were normalized to TBP and to GCN2 levels in the parental cell line. Each gene was analyzed in at least three independent experiments. The statistical analyses (unpaired t-test, Benjamini, Krieger and Yekutieli method) compared values between the depleted and overexpressing samples, ****p<0.0001, (D) p=0.0879 for CTTN, **p=0.0019 for GSN, *p=0.0111 for NET1, p=0.8585 for MAD2L1, ***p=0.0002 for SMC3, ***p=0.0009 for TUBGCP6, (E) **p=0.0096 for CTTN, **p=0.0025 for FLNA, **p=0.0088 for GSN, p=0.9396 for NET1, *p=0.0128 for MAD2L1, *p=0.0149 for SMC3, **p=0.0037 for NEK2, **p=0.0020 for TUBGCP6.

Article Snippet: Antibodies rabbit pericentrin 1:400 (Abcam, #ab4448); sheep α/β-tubulin 1:500 (Cytoskeleton Inc ATN02)

Techniques: Western Blot, Transfection, Stable Transfection, Transduction, Control

Journal: bioRxiv

Article Title: Analysis of patient data reveals novel cancer-relevant functions for GCN2/eIF2αK4

doi: 10.1101/2025.03.18.643965

Figure Lengend Snippet: A, B Progression through mitosis in the presence of GCN2i at the indicated concentrations was observed by live-cell imaging in A SiHa and B Caski cells. The time in mitosis is shown for each cell. Prophase was judged by the first frame showing chromatin condensation), anaphase was scored based on chromosome separation and mitotic slippage was scored based on decondensation of the DNA in the absence of chromosome separation. Brown Forsythe and Welch Anova test was performed on the total time in mitosis. SiHa 0 µM compared to 1 µM p=0.1236; 2 µM *p=0.0014; 3 µM ****p<0.0001. Caski 0 µM compared to 1 µM p=0.0551; 2µM and 3 µM **** p<0.0001. C Caski and SiHa cells were incubated in the absence (control) or presence (GCN2i) of GCN2i for 8 hours and fixed for immunofluorescence. Representative images of mitotic cells stained for tubulin (magenta), pericentrin (PCNT, cyan) and DNA (grey) are shown. Scale bars represent 5 µm. D High levels of GCN2 give no protection in starved cells. Hela cells transduced to overexpress GCN2 (GCN2 high ) and the parental cell line (Ctr) were grown in the presence of HFG at the indicated concentrations and observed in Incucyte. Data shown are from three independent experiments. Mean ± SEM are shown, non-linear regression, p=0.9867 for 0 nM, p=0.1607 for 25 nM, p=0.9012 for 50 nM, p=0.1994 for 75 nM, p=0.3283 for 100 nM. E High levels of GCN2 gives protection in cells exposed to MPS1i. Hela cells transduced to overexpress GCN2 (GCN2 WT ) and the parental cell line (Ctr) were grown in the presence of MPS1i at the indicated concentrations and observed in Incucyte. Data shown are from four independent experiments. Mean ± SEM are shown, non-linear regression, p=0.3393 for 0 µM, ****p<0.0001 for 3 µM, 4 µM, and 5 µM. F Protection in cells exposed to MPS1i is lost in cells expressing GCN2 RAXA . HeLa cells transduced to express siRNA-resistant GCN2 RAXA were transfected with GCN2-targeting siRNA (GCN2 RAXA ), grown in the presence of MPS1i at the indicated concentrations, and observed in Incucyte. Data shown are from four independent experiments. Mean and SEM are shown, non-linear regression, p= 0.5069 for 0 µM, p= 0.1346 for 3 µM, p=0.4104 for 4 µM, p=0.4254 for 5 µM. Note that the data shown for control cells are the same as those shown in .

Article Snippet: Antibodies rabbit pericentrin 1:400 (Abcam, #ab4448); sheep α/β-tubulin 1:500 (Cytoskeleton Inc ATN02)

Techniques: Live Cell Imaging, Incubation, Control, Immunofluorescence, Staining, Expressing, Transfection

Journal: bioRxiv

Article Title: Analysis of patient data reveals novel cancer-relevant functions for GCN2/eIF2αK4

doi: 10.1101/2025.03.18.643965

Figure Lengend Snippet: A Migration capacity correlates with GCN2 levels in hTert-RPE1 cells. hTert-RPE1 cells were transfected with GCN2-targeting siRNA for 48 h (siGCN2) or transduced to stably overexpress GCN2 (GCN2 high ). Cells were seeded without FBS into transwell chambers with 8 µM pore size and exposed to an FBS gradient for 16 hours. Migrated cells were counted after DAPI staining and normalized to seeding controls. Three independent experiments, mean and STDEV are shown. B Immunoblots of lysates of cells from the experiment shown in A. γ-tubulin is used as a loading control. C hTert-RPE1 cells were transfected with GCN2-targeting siRNA (siGCN2) or treated with 2 µM GCN2i (GCN2i). Cells were grown to confluence and wounds were created using a Wound Maker tool (Sartorius) and healing was monitored in Incucyte. Three (siGCN2) or five (GCN2i) independent experiments, mean ± SEM are shown, non-linear regression, ****p<0.0001 D HeLa cells treated with 2 µM GCN2i (GCN2i) or transduced to stably overexpress GCN2 (GCN2 high ) were seeded into Ibidi culture inserts and grown to confluence. After removal of the inserts the cells were observed by live-cell imaging. The number of cells migrating into the initial wound area is shown. Three (GCN2i) or four (GCN2 High ) independent experiments, mean and SEM are shown, non-linear regression, ****p<0.0001 E Representative images from an experiment shown in (D).

Article Snippet: Antibodies rabbit pericentrin 1:400 (Abcam, #ab4448); sheep α/β-tubulin 1:500 (Cytoskeleton Inc ATN02)

Techniques: Migration, Transfection, Stable Transfection, Pore Size, Staining, Western Blot, Control, Live Cell Imaging

Journal: bioRxiv

Article Title: Analysis of patient data reveals novel cancer-relevant functions for GCN2/eIF2αK4

doi: 10.1101/2025.03.18.643965

Figure Lengend Snippet: A , hTert-RPE1 and B, HeLa cells were grown to confluence and wounds were created using a Wound Maker tool (Sartorius) and healing was monitored in Incucyte. ISRIB or GCN2i was added to 200 nM or 2 µM, respectively. Four (hTert-RPE) or three (HeLa) independent experiments, mean and SEM are shown, non-linear regression, ****p<0.0001 C hTert-RPE1 cells transduced with GCN2-siR carrying the indicated mutations were transfected with control or GCN2-targeting siRNA for 48 h. Cells were seeded without FBS into transwell chambers with 8 µM pore size and exposed to an FBS gradient for 16 hours. Migrated cells were counted after DAPI staining and normalized to seeding controls, then to migration in cells transfected with control siRNA. Mean ± SEM are shown, results from at least three independent experiments. One-way Anova,****p<0.0001 siCtr versus siGCN2; **p=0.0022 siCtr versus GCN2 Wt siCtr; **p=0.0051 GCN2 Wt siGCN2 versus GCN2 RAXA siGCN2; **p=0.0025 GCN2 Wt siGCN2 versus GCN2 K619R siGCN2 D Knock-down efficiency and expression of the mutant transgenes in the experiments shown in C was tested by immunoblotting. γ-tubulin is shown as loading control. E hTert-RPE1 cells transduced with doxycycline-inducible siRNA-resistant GCN2 carrying the indicated mutations were transfected with control (siCtr) or GCN2-targeting siRNA (siGCN2) and grown to confluence in the presence of doxycycline. Wounds were created using a Wound Maker tool (Sartorius) and healing was monitored in Incucyte. ISRIB was added to 200 nM after wounding. Averages (lines) and SEM (bands) from six independent experiments are shown. Non-linear regression, **p=0.0028, ***p=0.0002, **** p<0.0001. F HeLa cells transduced with doxycycline-inducible siRNA-resistant GCN2 carrying the indicated mutations were transfected with control (siCtr) or GCN2-targeting siRNA (siGCN2), seeded into Ibidi culture inserts, and grown to confluence in the presence of doxycycline. After removal of the inserts the cells were observed by live-cell imaging. ISRIB was added to 200 nM after removal of the inserts. The number of cells migrating into the initial wound area is shown. Averages (lines) and SEM (bands) from three independent experiments are shown. Non-linear regression, ****p<0.0001, *p=0.0113, **p=0.0072, ***p=0.0006 G, H Metastatic potential (G) and penetrance (H) in metastatic cancers as a function of GCN2 mRNA levels. Data were downloaded from depmap.org and are based on a study by Jin et al (2020), which reported metastatic-potential profiling of ca. 500 human cancer cell lines derived from 21 types of solid tumour in immunodeficient murine models . Metastatic potential is calculated based on the mean cancer-cell numbers detected in the target organs. Metastatic penetrance refers to the proportion of mice displaying metastases with the given cell line .

Article Snippet: Antibodies rabbit pericentrin 1:400 (Abcam, #ab4448); sheep α/β-tubulin 1:500 (Cytoskeleton Inc ATN02)

Techniques: Transduction, Transfection, Control, Pore Size, Staining, Migration, Knockdown, Expressing, Mutagenesis, Western Blot, Live Cell Imaging, Derivative Assay