Fig 2: Markers for ASNase and APR-246 sensitivity in ALL cells.A p53 protein levels in untreated cells plotted against cell viability after 72 h treatment with APR-246. p53 levels were obtained by quantification of the western blot in B. Cell viability was assessed by resazurin (area under the curve [AUC]). See Table S5. Spearman correlation r = 0.12, p = 0.7. B Western blot of untreated ALL cell lines and a heat map indicating growth suppression as assessed by resazurin after 72 h of APR-246 treatment. C Analysis of data from the DepMap portal of the Broad Institute of 22 ALL cancer cell lines showing PRIMA-1 activity area under the curve (AUC) grouped into TP53 status. WT = wild type, Miss. Mut. = missense mutation, F.s. = frameshift, Nons. = nonsense, Del. = deletion D Analysis of data from the DepMap portal of the Broad Institute of 22 ALL cancer cell lines showing PRIMA-1 activity area under the curve (AUC) grouped into xCT/SLC7A11 mRNA expression below or above median. Unpaired t test, p = 0.08. E xCT protein levels in untreated cells plotted against cell viability after 72 h treatment with APR-246. xCT levels were obtained by quantification of the western blot in F. Cell viability was assessed by resazurin (area under the curve [AUC]). See Table S5. Spearman correlation r = 0.07, p = 0.9. F Western blot of untreated ALL cell lines and a heat map indicating growth suppression as assessed by resazurin after 72 h of APR-246 treatment (same as in B but in different order). G Total glutathione (GSH + GSSG) measured by enzymatic recycling assay in untreated ALL cell lines after 72 h in culture plotted against cell viability after 72 h of APR-246 treatment. Cell viability was assessed resazurin (area under curve [AUC]). Cell lines are specified in the figure legend box. See Table S5. Spearman correlation r = 0.2, p = 0.6 or excluding CCRF-SB r = 0.6, p = 0.1. H ASNS protein levels in untreated cells plotted against cell viability after 72 h treatment with APR-246. ASNS levels were obtained by quantification of the western blot in J. Cell viability was assessed by resazurin (area under the curve [AUC]). See Table S5. Spearman correlation r = 0.5, p = 0.1. I ASNS protein levels in untreated cells plotted against growth suppression after 72 h treatment with 0.003 U/ml ASNase. ASNS levels were obtained by quantification of the western blot in J. Growth suppression (%) was assessed by resazurin. See Table S4. Spearman correlation r = −0.7, p = 0.03. J Western blot of untreated ALL cell lines and a heat map indicating growth suppression as assessed by resazurin after 72 h of asparaginase (ASNase) treatment. Detailed information and n regarding growth suppression are given in Table S4. The same GAPDH control blot is shown in panels F and J since both xCT (F) and ASNS (J) were examined on the same blot. The blot was divided into an upper part which was probed with ASNS antibody and a lower part which was probed with xCT antibody and then re-probed with GAPDH antibody. Thus, the same GAPDH blot serves as control for both xCT and ASNS.

Fig 3: Endogenous NMD targets are mis-regulated in Smn +/− mice and SMA type I fibroblasts. (A) Protein lysate was collected from the spinal cord tissue of wt and Smn +/− mice. CARM1, UPF1 and SMN protein levels were assessed by western blotting. (B) Quantification of protein levels were gathered by normalizing to tubulin levels. Data are means +/− SEM (n = 3) (C) Total RNA was extracted from spinal cord tissue of wt and Smn +/− mice. RT-qPCR was used to measure the mRNA levels of targets which were normalized to Gapdh mRNA levels and presented as relative to wt target mRNA levels. Data are means +/− SEM (n = 3). (D,E) Fibroblast cell lines from two independent human SMA type I patients (GM00232, GM03813) were treated with Wortmannin (5 μM) for 6 h. Untreated fibroblast cell lines from a carrier (GM03815) were used as CTRL. Total RNA was isolated from the cell lines and three non-ASC-NMD target mRNA expression levels were measured by RT-qPCR: ARC, ASNS and ATF4 using GAPDH as a normalization control. Data are means +/− SEM (ARC, ATF4n = 6; ASNSn = 5).

Fig 4: NMD target stability and protein expression in Carm1 +/+ and Carm1 -/- MEFs. (A) Western blotting analysis and quantification shows levels of CARM1, UPF1 and NMD targets ASNS and ARC protein levels in Carm1 +/+ (wt) and Carm1 -/- MEFs. Tubulin was used as loading control. Data are means +/− SEM (n = 3). (B,C) Quantification of NMD transcripts Gadd45A and Asns mRNA half-lives normalized to Gapdh. Semi-logarithmic graph shows the decay rate of mRNAs calculated using formula: (x) = ln(0.5)/b, where b = slope obtained from the trendline formula: y = ne−bx. Carm1 +/+ and Carm1 -/- MEFs were pretreated with either a vehicle or Wortmannin (5 μM) and followed by treatment with actinomycin D (5 μg/ml) and RNA extraction at 0, 2, 4, 6 and 8 h. Quantification shows the means +/− SEM (n = 3) and data are presented as relative to zero time point.

Fig 5: Activating transcription factor 4 (ATF4) may regulate asparagine (Asn) metabolism by promoting asparagine synthetase (ASNS) expression in gastric cancer (GC). (A) Representative images for ASNS expression in tissue arrays of GC patients (n = 115). The final immunohistochemical (IHC) score was determined as the product of the staining intensity and the staining area. (B) Pearson’s correlation analysis for the levels of ATF4 and ASNS proteins in patients with GC measured by IHC (r = 0.494, p < 0.001). (C) A combination of the expressions of ATF4 and ASNS can more accurately predict overall survival (OS) in GC patients. Patients with high expressions of both ATF4 and ASNS showed the worst survival status. (D) ATF4 silencing significantly inhibited the level of ASNS in AGS and HGC27 cells. (E, G) Clone formation assay in medium supplemented or deprived of Asn after ATF4 knockdown. The results confirmed that the inhibition of cell clone formation under Asn deprivation was more significant in the ATF4 interference group. (F, H) Statistical tests comparing the inhibition of colony forming ability in the control and ATF4 interference groups under Asn-deprived conditions. Unpaired t-tests were used. **p < 0.01; ***p < 0.001; ns, not significant.