(A) Expression of the indicated eIF2α kinase was reduced in LNCaP cells using gene-specific siRNAs. Two different siRNAs were used for knockdown of each eIF2α kinase and compared to scrambled siRNA …
(A) LNCaP cells were transfected with the indicated gene-specific siRNAs or a scramble control and the protein levels of HRI, PERK, or PKR and p-eIF2α, total eIF2α, and ATF4 were measured by …
(A) Immunoblot analyses for 22Rv1, PC-3, and GCN2 KO clones. Protein lysates were analyzed by immunoblot to measure the levels of GCN2, ATF4, p-eIF2α, total eIF2α, or actin. Molecular weight markers …
(A) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured (N …
ATF4 expression was reduced in LNCaP, MR49F, C4-2B, or PC-3 cells using ATF4-specific siRNAs. Cell growth was measured in replicate wells (N = 5) for up to 6 days and is plotted as fold change (mean …
(A) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, …
H&E and IHC staining using p-GCN2-T899 antibody in prostatic needle biopsy specimens from patients with prostate cancer with Gleason scores 4 + 4 = 8 (top) or 4 + 5 = 9 (bottom). Non-malignant …
(A) Volcano plot illustrating log2 fold change in gene transcript levels with adjusted p value (−log10) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. …
(A) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative …
(A) Amino acid measurements of LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 8 hr. Bar graphs in the top panel show high abundance amino acids and the lower panel those with …
Bar graphs in the top panel show high abundance amino acids and the lower panel highlights those with lower levels. Statistical significance was determined using an unpaired two-tailed t-test. Error …
LNCaP cells were treated with GCN2iB (2 µM) or vehicle control (DMSO) in standard growth media, media supplemented with non-essential amino acids (NEAA), supplemented with essential amino acids …
(A) LNCaP, MR49F, C4-2B, 22Rv1, or PC-3 cells were transfected with GCN2 siRNA, ATF4 siRNA, or scrambled siRNA control for 48 hr and subjected to cell cycle analysis as described in the Materials …
(A) LNCaP cells were treated with 2 µM GCN2iB or vehicle (DMSO) control in the absence or presence of EAA for 8 hr and tRNAHis charging levels were determined by qRT-PCR as described in the …
(A) MR49F cells were treated with GCN2iB (2 µM) or vehicle control (DMSO) for 96 hr in normal growth media, growth media supplemented with essential amino acid (EAA), or growth media supplemented …
(A) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x-axis. Significantly depleted genes (p ≤ 0.05) in …
LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) …
(A) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), (B) cultured in …
(A) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using …
Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of …
(A) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is …
(A) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) …
(A) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), …
(A) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in Figure 6G. Statistical significance was …
(A) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. …
Male NSG mice were injected subcutaneously with LNCaP (N = 5) (A) or 22Rv1 (N = 4) (B) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 (N = 5) (C). …
Male NSG mice were injected subcutaneously with LNCaP (N = 5) or 22Rv1 (N = 4) cells, or implanted with tumor fragments from an androgen-sensitive tumor TM00298 (N = 5) or the castration-resistant …
Immunoblot analysis of 22Rv1 (A) or TM00298 (B) tumors from mice treated with GCN2iB as described in Figure 7. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The …
(A) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue (N = 10) …
Statistical analysis for growth assays.
Normalized read counts and analysis of RNA-seq data.
Normalized read counts and analysis of Charge-seq data.
CRISPR screen analysis of depleted genes and single guide RNAs (sgRNAs).
Supplementary tables.
Western blot source data for main figures, including all individual uncropped western blot images.
Western blot source data for Figure 1—figure supplement 1 and Figure 1—figure supplement 2, including all individual uncropped western blot images.
Western blot source data for Figure 1—figure supplement 3, including all individual uncropped western blot images.
Western blot source data for Figure 1—figure supplement 5, Figure 3—figure supplement 2, Figure 3—figure supplement 4, Figure 4—figure supplement 3, and Figure 6—figure supplement 2, including all individual uncropped western blot images.
Western blot source data for Figure 6—figure supplement 3, Figure 6—figure supplement 4, and Figure 7—figure supplement 2, including all individual uncropped western blot images.