p junb  (Cell Signaling Technology Inc)

Journal: eLife

Article Title: Neutral amino acid transporter SLC38A2 protects renal medulla from hyperosmolarity-induced ferroptosis

doi: 10.7554/eLife.80647

Figure Lengend Snippet: ( A ) Inhibitory effect of hyperosmolarity on the mTORC1 signalling pathway. mIMCD3 cells were incubated with hyperosmotic solutions for 12 h. Western blotting analysis showed that hyperosmolarity suppressed the total and phosphorylated S6 and 4E-BP1 expression in a dose-dependent manner. ( B and C ) MTT assay results showing that rapamycin ( B ) or torin-1 ( C ) sensitised mIMCD3 cells to erastin-induced ferroptosis under hyperosmolality. mIMCD3 cells were treated with rapamycin or torin-1 at various doses for 24 h. During the last 12 h, the cells were treated with or without erastin (10 μM) in the presence or absence of hyperosmolarity (600 mOsm). n=8. ( D ) The mTORC1 inhibitors, rapamycin and torin-1, reduced the activity of glutathione peroxidase (GPx) activity. The cells were treated with rapamycin (10 nM) or torin-1 (10 nM) for 24 h, with or without hyperosmotic treatment in the final 12 h. n=6. ( E ) Cytometry assay showing that rapamycin and torin-1 significantly increased the levels of lipid reactive oxygen species (ROS). n=3. ( F ) Western blotting assay results demonstrating that SLC38A2 overexpression reversed hyperosmolarity-suppressed expression of total and phosphorylated S6 and 4E-BP1. The cells were infected with Ad-SLC38A2 or Ad-GFP for 36 h followed by hyperosmotic stress for 12 h. ( G–H ) MTT assay results showing that rapamycin ( G ) and torin-1 ( H ) completely abolished the protective effect of SLC38A2 in mIMCD3 cells under hyperosmolarity. After being infected with the adenoviruses, the cells were treated with rapamycin (10 nM) or torin-1 (10 nM) for 24 h with or without hyperosmolarity in the last 12 h. n=9–12. Data are means ± SEM; two-way ANOVA tests for B and C; one-way ANOVA tests for D, E, G, and H. See numerical source data and uncropped western blot images in . Figure 9—source data 1. Numerical and uncropped western blot source data for .

Article Snippet: Antibodies against NF-κB (Cat#8242), p-NF-κB (Ser 536 , Cat#3033), IκBα (Cat#9242), p-IκBα (Ser , Cat#2859), rpS6 (Cat#2217), p-S6 (Ser 235/236 , Cat#4858), 4E-BP1 (Cat#9644), p-4E-BP1 (Thr 37/46 , Cat#2855), GPX4 (Cat#52455), caspase-3 (Cat#9662), and cleaved caspase-3 (Cat#9664) were purchased from Cell Signalling Technology.

Techniques: Incubation, Western Blot, Expressing, MTT Assay, Activity Assay, Cytometry, Over Expression, Infection

Journal: Oncogene

Article Title: The translational repressor 4E-BP1regulates RRM2 levels and functions as a tumor suppressor in Ewing Sarcoma Tumors

doi: 10.1038/s41388-020-01552-0

Figure Lengend Snippet: Inhibition of mTORC1/2 activates 4E-BP1, blocks protein synthesis, and reduces the level of the RRM2 protein. (A, B, C) Ewing sarcoma cell lines were treated with TAK-228 (1 μM) or temsirolimus (1 μM) for 6 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (D, E) Ewing sarcoma cell lines were treated with AZD2014 (1 μM) for 6 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (F) Sarcoma cell lines were treated with TAK-228 (1 μM) for 6 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (G) EW8 and TC71 cells were treated with TAK-228 (100 nM), AZD1775 (100 nM), or the combination of the drugs for 6 h. (H, I) EW8 and A673 cells were treated for 72 h with a combination of TAK-228 and AZD1775, or TAK-228 in combination with prexasertib. Survival was assayed by Cell-Titer-Glo and each experiment was repeated 2 times. Loewe matrix plots for drug cooperativity are shown. Protein loading for all of the immunoblots was normalized using cell number.

Article Snippet: Antibodies to the following proteins were used in the immunoblots: phospho-Histone-139 H2A.X (Cell Signaling, #9718, 1:1000), phospho-Chk1-345 ( Cell Signaling, #2348, 1:1000), Chk1 (Cell Signaling, #2360, 1:1000), puromycin (Millipore, #AF488, 1:2000), 4E-BP1 (Cell Signaling, #9644, 1:1000), PARP (Cell Signaling, #9532, 1:1000), p-4E-BP1-37/46 (Cell Signaling, #2855, 1:1000), p-4E-BP1-65 (Cell Signaling, #9451, 1:1000), p-4E-BP1-70 (Cell Signaling, #9455, 1:1000), RRM1 (Cell Signaling, #8637, 1:1000), RRM2 (Santa Cruz, #398294, 1:500), FLAG (Sigma, F1804, 1:1000), actin (Proteintech, 60008-1, 1:5000), and tubulin (Proteintech, 66031-1, 1:2000).

Techniques: Inhibition, Labeling, Western Blot

Journal: Oncogene

Article Title: The translational repressor 4E-BP1regulates RRM2 levels and functions as a tumor suppressor in Ewing Sarcoma Tumors

doi: 10.1038/s41388-020-01552-0

Figure Lengend Snippet: The CRISPR/Cas9-mediated knockout of 4E-BP1 in Ewing sarcoma cell lines rescues the effects of mTORC1/2 inhibitors on protein synthesis. (A) Immunoblot showing 4E-BP1 expression level after CRISPR/Cas9-mediated gene knockout. This immunoblot reflects 4E-BP1 levels in the bulk cell population prior to single cell cloning. (B, C) The 4E-BP1-KO and parental cells were treated with TAK-228 (1 μM) for 3 h or 6 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (D) The 4E-BP1-KO and parental cells were treated with AZD2014 (1 μM) for 6 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (E, F) Dose response curves for the 4E-BP1-KO and parental (4E-BP1-WT) cells. Cell viability was assessed 72 h after drug addition using the AlamarBlue assay. Error bars represent the mean ± SD of three technical replicates. The results are representative of two independent experiments. (G) RT-qPCR for RRM2 mRNA in EW8 and TC71 cells treated with TAK-228 (100 nM) for 24 h. Protein loading for all of the immunoblots was normalized using cell number.

Article Snippet: Antibodies to the following proteins were used in the immunoblots: phospho-Histone-139 H2A.X (Cell Signaling, #9718, 1:1000), phospho-Chk1-345 ( Cell Signaling, #2348, 1:1000), Chk1 (Cell Signaling, #2360, 1:1000), puromycin (Millipore, #AF488, 1:2000), 4E-BP1 (Cell Signaling, #9644, 1:1000), PARP (Cell Signaling, #9532, 1:1000), p-4E-BP1-37/46 (Cell Signaling, #2855, 1:1000), p-4E-BP1-65 (Cell Signaling, #9451, 1:1000), p-4E-BP1-70 (Cell Signaling, #9455, 1:1000), RRM1 (Cell Signaling, #8637, 1:1000), RRM2 (Santa Cruz, #398294, 1:500), FLAG (Sigma, F1804, 1:1000), actin (Proteintech, 60008-1, 1:5000), and tubulin (Proteintech, 66031-1, 1:2000).

Techniques: CRISPR, Knock-Out, Western Blot, Expressing, Gene Knockout, Cloning, Labeling, Alamar Blue Assay, Quantitative RT-PCR

Journal: Oncogene

Article Title: The translational repressor 4E-BP1regulates RRM2 levels and functions as a tumor suppressor in Ewing Sarcoma Tumors

doi: 10.1038/s41388-020-01552-0

Figure Lengend Snippet: The inducible expression of constitutively-active 4E-BP1 reduces the level of the RRM2 protein and protein synthesis in sarcoma cells. (A) The doxycycline-inducible 4E-BP1-Ala cell lines were treated with doxycycline for 24 h. Cellular lysates were then collected for immunoblotting. (B) RT-qPCR for RRM2 mRNA in the 4E-BP1-Ala cell lines treated with doxycycline or DMSO for 24 h. (C) The doxycycline-inducible 4E-BP1-Ala cell lines were treated with doxycycline for 24 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (D) The 4E-BP1-Ala cell lines were treated with doxycycline or DMSO for 72 h. Cell viability was then quantified using the AlamarBlue assay. Error bars represent the mean ± SD of three technical replicates. The results are representative of two independent experiments. (E) The 4E-BP1-Ala cell lines were treated with doxycycline or DMSO for 14 days and then the number of colonies were counted. Error bars represent the mean ± SD of three technical replicates. (F) TC71 cells with doxycycline-inducible expression of 4E-BP1-Ala were implanted subcutaneously (Day 0) in NCr mice. Mice were then fed standard chow (n=8) or doxycycline-containing chow (n=8). Tumor size was quantified every 2-3 days using caliper measurements. The plot shows the tumor growth for the individual mice, as well as the mean tumor volume (dotted line) for the mice receiving standard or doxycycline-containing chow. (G) HT1080, RD, and U2OS doxycycline-inducible 4E-BP1-Ala cell lines were treated with doxycycline for 24 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. Protein loading for all of the immunoblots was normalized using cell number.

Article Snippet: Antibodies to the following proteins were used in the immunoblots: phospho-Histone-139 H2A.X (Cell Signaling, #9718, 1:1000), phospho-Chk1-345 ( Cell Signaling, #2348, 1:1000), Chk1 (Cell Signaling, #2360, 1:1000), puromycin (Millipore, #AF488, 1:2000), 4E-BP1 (Cell Signaling, #9644, 1:1000), PARP (Cell Signaling, #9532, 1:1000), p-4E-BP1-37/46 (Cell Signaling, #2855, 1:1000), p-4E-BP1-65 (Cell Signaling, #9451, 1:1000), p-4E-BP1-70 (Cell Signaling, #9455, 1:1000), RRM1 (Cell Signaling, #8637, 1:1000), RRM2 (Santa Cruz, #398294, 1:500), FLAG (Sigma, F1804, 1:1000), actin (Proteintech, 60008-1, 1:5000), and tubulin (Proteintech, 66031-1, 1:2000).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Labeling, Alamar Blue Assay

Journal: Oncogene

Article Title: The translational repressor 4E-BP1regulates RRM2 levels and functions as a tumor suppressor in Ewing Sarcoma Tumors

doi: 10.1038/s41388-020-01552-0

Figure Lengend Snippet: Integrated model for the regulation of RRM2 protein levels by the mTORC1/2, ATR-CHK1, and WEE1 pathways. In previous work, we found that the inhibition of ATR-CHK1-WEE1 signaling enhances the CDK-mediated degradation of RRM2 . In the current work, we showed that the inhibition of mTORC1/2 blocks the synthesis of the RRM2 protein via the activation of 4E-BP1.

Article Snippet: Antibodies to the following proteins were used in the immunoblots: phospho-Histone-139 H2A.X (Cell Signaling, #9718, 1:1000), phospho-Chk1-345 ( Cell Signaling, #2348, 1:1000), Chk1 (Cell Signaling, #2360, 1:1000), puromycin (Millipore, #AF488, 1:2000), 4E-BP1 (Cell Signaling, #9644, 1:1000), PARP (Cell Signaling, #9532, 1:1000), p-4E-BP1-37/46 (Cell Signaling, #2855, 1:1000), p-4E-BP1-65 (Cell Signaling, #9451, 1:1000), p-4E-BP1-70 (Cell Signaling, #9455, 1:1000), RRM1 (Cell Signaling, #8637, 1:1000), RRM2 (Santa Cruz, #398294, 1:500), FLAG (Sigma, F1804, 1:1000), actin (Proteintech, 60008-1, 1:5000), and tubulin (Proteintech, 66031-1, 1:2000).

Techniques: Inhibition, Activation Assay