Fig 2: Cell-type-specific ribosome profiling identifies novel prion disease induced changes.(a) Table displaying the number of changes in the different cell types at the terminal stage and how many of them are preferentially translated genes (PTGs). Shown are numbers of genes that change either in the ribosome profiling (RP) or the RNAseq or in both datasets. (d) Boxplots displaying terminal RNA expression changes in the hippocampus of all expressed genes and PTGs. (c) Scatterplot comparing prion-induced RNA expression changes in the hippocampus and translational changes in GFAP+ cells. GFAP PTGs and genes that change only in the RP but not the RNAseq dataset are indicated. (d) Scatterplot comparing prion-induced RNA expression changes in the hippocampus and translational changes in Cx3cr1+ cells. Cx3cr1 PTGs and genes that change only in the RP but not the RNAseq dataset are indicated. (e) Bars represent the translation (log2 average of normalized counts per gene; DESeq2 output) and translational change (log2FC; DESeq2 output) in the different cell types. Shown are genes that are enriched in astrocytes (Glul and Gfap) and microglia (Aif1 encoding for IBA1, and Mmp9). Significant changes (Benjamini Hochberg adjusted p value < 0.05; derived from DESeq2 analyses) are marked with an asterisk. (f) Western blot and its quantification showing protein levels of astrocyte (GLUL and GFAP) and microglia (IBA1 and MMP9) enriched genes in control (ctrl) and terminal prion disease (PrD) samples (*p<0.05; **p<0.01; ***p<0.001; two-tailed t test comparing terminal PrD vs ctrl samples; error bars: standard deviation). (g) Western blot and its quantification showing neuronal protein levels (NeuN in cortex and hippocampus, PV in whole brain) in ctrl and terminal PrD samples (ns = not significant; two-tailed t test comparing terminal PrD vs ctrl samples; error bars: standard deviation).

Fig 3: Decreased GS expression in HCC827 cells is associated with acquired resistance to gefitinib.a According to the MTT assays, HCC827 GR cells became resistant to gefitinib after chronic and repeated exposure to increasing doses of gefitinib compared to HCC827 cells, which were sensitive to gefitinib. b, c Changes in GLUL mRNA expression levels were quantified by qRT-PCR (b), and GS protein levels were examined by western blotting (c) to compare the levels between HCC827 and HCC827 GR cells after exposure to the gefitinib or control treatment for 48 h. The bars shown are normalized to the GAPDH control and represent the mean ± SD of triplicate samples. d Glutamine levels in HCC827 and HCC827 GR cells were assessed after a 24-h exposure to 5 µM and 5 nM gefitinib, respectively. e–g After exposing HCC827 and HCC827 GR cells to 0, 0.1, 1, and 10 µM gefitinib for 72 h, the intracellular ATP (e), GSH (f), and ROS levels (g) were measured using the ATP determination kit, the GSH-Glo glutathione assay kit, and the DCFH-DA reagent, respectively. The gefitinib treatment significantly reduced the normalized ATP and GSH levels in HCC827 cells in a dose-dependent manner. Compared to the stable ROS level in HCC827 cells, the total ROS level in HCC827 GR cells was reduced, indicating scavenging. The data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, two-tailed Student’s t-test

Fig 4: Expression of GLUL in A549 cells sensitizes them to the gefitinib treatment and decreases cell motility, whereas the loss of GLUL expression in PC-9 cells increases resistance to gefitinib treatment and increases cell motility.a qRT-PCR and western blotting were used to assess the GLUL mRNA level and the GS protein level, respectively, to identify the GLUL knock-in efficacy in A549 cells and the GLUL knockout efficacy in PC-9 cells. The bars shown are normalized to the GAPDH control and represent the mean ± SD of triplicate samples. b MTT assays detected the cell growth inhibition ratios following the gefitinib treatment in GLUL-expressing A549 cells and GLUL knockout PC-9 cells. c Based on the transwell assay, significantly fewer A549-GLUL cells invaded the membrane than A549 cells, and the 24-h gefitinib treatment further suppressed the invasion of A549-GLUL cells. In contrast to A549-GLUL cells, the gefitinib treatment did not inhibit the invasion of A549 cells. d Scratch wound-healing assays showed that the knockout of GLUL in PC-9 cells resulted in a decrease of the ability of cells to close a wound after the 24-h treatment with gefitinib

Fig 5: GLUL and GS levels were upregulated in gefitinib-sensitive cells in response to the gefitinib treatment. Gefitinib-resistant cells lack GLUL expression or exhibit no significant changes following the gefitinib treatment.a After separately exposing A549 and PC-9 cells to 20 µM and 20 nM gefitinib, respectively, for 48 h, DNA microarray scatter plots were prepared to reveal the expression of activation-induced genes in gefitinib-treated cells compared with that in the corresponding control cells. Each point represents a gene; the red points indicate genes that significantly upregulated in gefitinib-treated cells (ratio ≥ 2-fold, p < 0.05), whereas the green points indicate genes that were significantly downregulated (ratio ≤ 0.5-fold, p < 0.05) in response to the gefitinib treatment. The black points represent genes for which the signal intensity ratio was between 0.5 and 2, indicating that gefitinib treatment had no obvious effect on these genes. b A scheme displays the relationships between the differentially expressed genes in A549 and PC-9 cells. The genes related to glutamine metabolism are listed. The red- and green-colored genes represent increased and decreased gene expression, respectively, in gefitinib-treated cells. c Changes in the mRNA expression levels of seven important genes (GGCT, GLUL, MGST2, NADSYN1, ODC1, RRM1, and RRM2) in A549 and PC-9 cells in response to the 48-h gefitinib treatment are shown. The data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, two-tailed Student’s t-test. d Western blot detection of the levels of the GS protein in A549 and PC-9 cells after treatment with 20 µM and 20 nM gefitinib, respectively, for 48 h. e, f Changes in GLUL mRNA expression levels were quantified by qRT-PCR (e), and the GS protein levels were examined by western blotting (f) in cells treated with gefitinib for 48 h and the corresponding control cells. The bars shown are normalized to the GAPDH control and represent the mean ± SD of triplicate samples