Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Phagolysosomes do not undergo exocytosis but fragment. (A) 1 h after phagocytosis of IgG-opsonized beads, RAW cells were treated with 10 µM ionomycin in the presence of 1.2 mM of Ca 2+ , and live-cell DIC images were acquired at the indicated time points. Scale bars: 10 µm. (B) Number of latex beads per cell at the indicated treatment times, normalized to the number of beads/cell observed at the onset of the treatment (T 0 ). Data are mean ± SEM of 3 independent experiments, where 10 cells were measured in each experiment. (C) RAW cells expressing PLCδ1-PH-GFP and containing IgG-opsonized beads in phagosomes were treated with 10 µM ionomycin or DMSO (control) in the presence of 1.2 mM of Ca 2+ . Images were acquired before or after 10 min of treatment. Asterisks correspond to the positions of the beads, as shown in the insets. Scale bars: 10 µm. (D) Live-cell imaging time series of IgG-aggregated fluorescent 0.1-µm latex beads after 2-h phagocytosis by RAW cells. Beads are depicted in a rainbow scale. Red and blue correspond to the highest and lowest florescence intensity levels, respectively. The smaller panels show the far red and DIC channels for the first frame. The cell contour is delineated with white dashes. See corresponding . Scale bars: 5 µm. (E) Macrophages were assessed for exocytosis of digested phagosomal contents. RAW macrophages internalized mRFP1-labeled E. coli for 1 h and then were incubated for the indicated time points. TCA precipitates of cell media and cell lysates were probed for mRFP1, detecting a native and a cleaved mRFP1 product that accumulated over time. GAPDH was used as a loading control and to detect cell lysis. (F) Quantification of cleaved mRFP1 levels normalized to GAPDH in media and within cells. Data are shown as mean ± SD of six independent experiments. Conditions were compared using one-way ANOVA and Holm-Sidak’s post hoc test (*, P < 0.05).

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Expressing, Control, Live Cell Imaging, Labeling, Incubation, Lysis

Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Fragmentation of the phagolysosome requires cargo degradation and the cytoskeleton. (A, C, E, and G) Macrophages engulfed E. coli for 15 min, chased for 25 min, and treated with either Con A and NH 4 Cl (A), protease inhibitor cocktail (C), microtubule inhibitors (E), actin inhibitors (G), or vehicle control (DMSO). Cells were fixed after 1 or 4 h after phagocytosis and stained with an anti– E. coli antibody whose fluorescence is displayed in rainbow scale. Dashed lines show cell contours. Scale bars: 5 µm. (B, D, F, and H) Total volume of PDVs per cell. Data are mean ± SEM of 3 independent experiments with 25 cells quantified per condition of an experiment and compared by one-way ANOVA with Tukey’s test. Conditions labeled with different letters (a–d) are statistically different (P < 0.05).

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Protease Inhibitor, Control, Staining, Fluorescence, Labeling

Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Clathrin is necessary for the resolution of the phagolysosome. (A) Imaging of RAW cells 4 h after phagocytosis of mCherry- Lp (rainbow scale). Pitstop was added 10 min before imaging. See corresponding . Scale bars: 5 µm. (B) Quantification of the volume of phagosomes and PDVs over time. Volumes were normalized to T 0 for each treatment. Data are mean ± SEM of three independent experiments. *, P < 0.05 indicates that the regressions are significantly different. (C) RAW cells engulfed mRFP1– E. coli for 15 min, chased for 25 min, and treated with Pitstop or ikarugamycin. Cells were fixed 1 or 4 h after phagocytosis and immunostained against E. coli (rainbow scale). Scale bars: 5 µm. (D) Volume of PDVs per cell for experiments shown in C. Data are mean ± SEM of 3 independent experiments with 25 cells quantified per condition of an experiment, and results were tested by one-way ANOVA with Tukey’s test. Different letters indicate results are statistically different (P < 0.05). (E) RAW cells expressing Mito-mCherry-FRB and GFP-FKBP-CLC were treated with rapamycin to induce knocksideways (KSW) of the clathrin light chain or DMSO (Ctrl) and allowed to internalize E. coli . Cells were fixed 3 h after phagocytosis, stained for E. coli , and imaged. Scale bars: 10 µm. (F) Number of fragments stained with anti– E. coli antibodies in control and rapamycin-treated cells. Data are mean ± SEM of three independent experiments and statistically tested using an unpaired t test (*, P < 0.05). Dashed lines show cell contours (A, C, and E).

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Imaging, Expressing, Staining, Control

Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Dynamin inhibition blocks phagosome fragmentation. (A and C) 40 min after phagocytosis of mRFP1– E. coli , RAW cells were treated with the dynamin inhibitors dyngo-4a, dynole 34-2, or vehicle (DMSO); fixed at the indicated time points; and immunostained for E. coli . E. coli fluorescence labeling is shown in rainbow scale. Red and blue are the highest and lowest intensity levels, respectively. Dashed lines indicate the boundary of the cells. Scale bars: 5 µm. (B and D) The total volume of PDVs per cell for the indicated treatments. Data are mean ± SEM of sample of 25 cells from 3 independent experiments. Treatments were compared statistically using a one-way ANOVA with Tukey’s post hoc test. Conditions with different letters (a–c) indicate statistically significant difference (P < 0.05). (E) Macrophages treated with dynole 34-2 or DMSO (vehicle) internalized Alexa Fluor 546–labeled transferrin for 10 min before fixation. White dashes indicate cell boundaries. Scale bars: 30 µm.

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Inhibition, Fluorescence, Labeling

Journal: Mucosal Immunology

Article Title: Spatial proteomics revealed a CX 3 CL1-dependent crosstalk between the urothelium and relocated macrophages through IL-6 during an acute bacterial infection in the urinary bladder

doi: 10.1038/s41385-020-0269-7

Figure Lengend Snippet: Mice were infected with UPEC and analyzed 1-day post-infection. a The density of F4/80 + macrophages, in which macrophages were either deficient ( LysM cre/+ gp130 fl/fl ) or competent ( LysM +/+ gp130 fl/fl ) in IL-6 receptor signaling, was calculated by SCHNELL on immunofluorescent images. b Detailed expression intensities [au] of the chemokines indicated within the urothelium by MALDI-MSI. c The urothelial density of F4/80 + macrophages was determined by immunofluorescence microscopy after topical treatment with CCR1 (control: DMSO), CCR3 (control: IgG2b) and CCR5 (control: DMSO) inhibitors. d – g Bladder tissue sections from fractalkine receptor competent ( Cx 3 cr1 +/gfp ) and -deficient ( Cx 3 cr1 gfp/gfp ) mice were stained with F4/80 (red) and acquired by fluorescence microscopy. The connective tissue is shown on the top panel ( d , e ), and urothelium on the bottom panel ( f , g ). The scale bars in the first column indicate 200 µm, second column 50 µm. e , g The density of F4/80 + macrophages was calculated by SCHNELL on the immunofluorescent images (Representative images shown in d , f ). * p < 0.05, ** p < 0.01. Data are means ± SEM.

Article Snippet: DMSO, control for CCR1 and CCR5 , PanReac AppliChem , Cat #A3672,0250.

Techniques: Infection, Expressing, Immunofluorescence, Microscopy, Control, Staining, Fluorescence

Journal: Mucosal Immunology

Article Title: Spatial proteomics revealed a CX 3 CL1-dependent crosstalk between the urothelium and relocated macrophages through IL-6 during an acute bacterial infection in the urinary bladder

doi: 10.1038/s41385-020-0269-7

Figure Lengend Snippet:

Article Snippet: DMSO, control for CCR1 and CCR5 , PanReac AppliChem , Cat #A3672,0250.

Techniques: Control, Inhibition, Immunofluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Software, Imaging, Light Microscopy

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( 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 control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and is plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001. ( B ) LNCaP cells were transfected with two different siRNAs targeting GCN2 or a scramble siRNA control and cell lysates were prepared and immunoblotted for the indicated proteins. Molecular weight markers are shown in kilodaltons. The relative levels of p-eIF2α normalized to total eIF2α compared to scramble siRNA control are indicated. ( C ) Expression of GCN2 was knocked-down in LAPC-4, C4-2B, MR49F, 22Rv1, or PC-3 cells using two different siRNAs and compared to scrambled siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way ANOVA as described in ; ***p ≤ 0.001, ****p ≤ 0.0001. ( D ) LNCaP cells were treated with indicated concentrations of GCN2iB and cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤0.0001. ( E ) LNCaP cells were treated with GCN2iB (2 µM) or DMSO control for 24 hr and protein lysates were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin as indicated. Relative levels of p-eIF2α normalized to total eIF2α are shown. ( F ) Levels of p-GCN2 were measured in prostate tumor microarrays (Biomax PR1921b and PR807c) using immunohistochemistry (IHC). Staining for p-GCN2-T899 from non-malignant ( N = 33) and malignant PCa tissue ( N = 88) from patients >50 years old was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing p-GCN2-T899 staining of normal and malignant prostate tissues are shown. Scale bars shown are 200 µm (main image) and 20 µm (insert).

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Expressing, Standard Deviation, Transfection, Molecular Weight, Western Blot, Immunohistochemistry, Two Tailed Test, Staining

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( 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 = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Cell Culture, Standard Deviation, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( 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, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Western Blot, Molecular Weight, Transfection, Expressing, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Expressing, Western Blot, Molecular Weight, Cell Culture, Two Tailed Test

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( 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 levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Two Tailed Test, Standard Deviation, Western Blot

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( 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 lower levels. The heat map on the right shows fold change in amino acid abundance for each biological replicate of GCN2iB-treated LNCaP cells versus the vehicle with the scale showing the highest fold change in yellow and lowest in purple. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. ( B ) LNCaP cells were treated with vehicle, GCN2iB (2 µM), vehicle + essential amino acids (EAA), or GCN2iB (2 µM) + EAA, and cell growth was measured for up to 6 days. Error bars indicate SD ( N = 5). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( C ) Cell cycle analyses of LNCaP cells treated with vehicle, GCN2iB (2 µM), vehicle + EAA, or GCN2iB (2 µM) + EAA for 48 hr. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 3); ***p ≤ 0.001, ****p ≤ 0.0001. ( D ) Genome-wide tRNA charging analysis (CHARGE-seq) of LNCaP cells treated with vehicle (DMSO), GCN2iB (2 µM), or GCN2iB (2 µM) + EAA for 8 hr. The tRNA charging ratio is shown as a bar graph with fold change compared to vehicle. Only tRNA isoacceptors measured in LNCaP cells are shown. Error bars indicate SD ( N = 4). ( E ) tRNA charging percentage for tRNA His in LNCaP cells treated with vehicle, GCN2iB, or GCN2iB + EAA. Statistical significance was determine using a one-way ANOVA with Tukey’s multiple comparisons ( N = 4); ***p ≤ 0.001, ****p ≤ 0.0001. ( F ) LNCaP cells were treated with vehicle, GCN2iB (2 µM), GCN2iB (2 µM) + EAA, or GCN2iB (2 µM) combined with the indicated individual amino acids. Cell growth was measured at 4 days in triplicate wells ( N = 3). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD; ****p ≤ 0.0001. ( G ) Cell cycle analysis of LNCaP cells were treated with vehicle, GCN2iB (2 µM), GCN2iB (2 µM) + histidine (200 µM), or with media lacking histidine for 48 hr. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 3); ***p ≤ 0.001, ****p ≤ 0.0001. ( H ) LNCaP cells were cultured in normal media, media supplemented with EAA mix, or media supplemented with histidine (200 µM) for 24 hr. Lysates were analyzed by Immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin. Molecular weight markers are presented in kilodaltons for each immunoblot panel. The relative levels of p-eIF2α normalized to total eIF2α compared to normal media (NM) control are indicated.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Two Tailed Test, Standard Deviation, Genome Wide, Cell Cycle Assay, Cell Culture, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: 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 (EAA), or supplemented with histidine (200 µM), and cultured for up to 6 days. Cell growth was quantitated using CellTiter-Glo as described in the Materials and methods and is presented as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Cell Culture, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle (DMSO) control in the absence or presence of EAA for 8 hr and tRNA His charging levels were determined by qRT-PCR as described in the Materials and methods. ( B ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the absence or presence of histidine supplementation. Puromycin (1 µM) was added to culture media 15 min prior to lysate preparation and puromycin incorporation was measured by immunoblot analysis using anti-puromycin antibody. Puromycin incorporation was quantified from replicate samples and is shown normalized to the vehicle control levels. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 3); ns, p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( 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 with individual amino acids as indicated. Cell growth was quantified using CellTiter-Glo as described in the Materials and methods and is presented normalized to the vehicle control group. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO cells were cultured for 96 hr in normal growth media, growth media supplemented with EAA, or growth media supplemented with individual amino acids as indicated and cell growth was similarly measured as described in A . Statistical significance in panels A and B was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 3); ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Cell Culture, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( 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 or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Standard Deviation, Transfection, Stable Transfection, Plasmid Preparation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( 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 standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Quantitative RT-PCR, Cell Culture, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( 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 antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Standard Deviation