Journal: Journal of Biological Chemistry

Article Title: A Self-defeating Anabolic Program Leads to β-Cell Apoptosis in Endoplasmic Reticulum Stress-induced Diabetes via Regulation of Amino Acid Flux

doi: 10.1074/jbc.m113.466920

Figure Lengend Snippet: FIGURE 6. Attenuation of translational recovery during ER stress in Min6 cells promotes survival. A, [35S]Met/Cys incorporation into proteins in cells treated with Tg and Sal003 for the indicated times. Sal003 was added 6 h after initiation of Tg treatment as indicated by the scheme. B, Western blot analysis of the indicated proteins from cells treated with Tg and Sal003 (7.5 M for the last 6 or 12 h of Tg treatment). Antibodies against LRS and XPOT were from Abcam;othersarelistedinFig.1.C,LeuandMeAIBuptakeincellstreatedwith Tg and Sal003 (7.5 M) for the indicated times. D, cells were treated as indi- cated and apoptosis was assessed by measuring the percentage of sub-G1 cells by flow cytometry of propidium iodide-stained cells. Leu starvation was performed by replacing the media after 6 h of Tg treatment with Tg-contain- ing Leu-free medium. D-Leu (40 mM) or Sal003 (7.5 M) was added for the last 6 h of Tg treatment. Significant differences (p 0.01) from untreated cells (*) or the appropriate Tg-treated cells (**) are indicated. Error bars, S.E.

Article Snippet: Sal003 was from Tocris.

Techniques: Western Blot, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Regulation of RNA methylation by therapy treatment, promotes tumor survival

doi: 10.1101/2023.05.19.540602

Figure Lengend Snippet: Western blots of MCF7 cells treated with A. poly I:C to induce eIF2α phosphorylation (2ug/ml). B. m6A dot blot analysis with poly I:C treatment. C. Treatment of hormone positive patient sample BT29 and BT30 cells with Sal003 (10uM) phosphatase inhibitor to retain eIF2α phosphorylation. D. Western blot of METTL3, METTL14 and ATF4 levels that do not increase when 500nM doxorubicin-treated patient sample cells that are also co-treated with 5uM Trazodone, or with E. with 1uM ISRIB, to override the effect of eIF2α phosphorylation. F. qPCR analysis of METTL3 and METTL14 RNA levels in 500nM doxorubicin (1day) treated MCF7 cells, compared to untreated cells. Polysome fractionation followed by qPCR analysis of polysome fractions G. for METTL3 mRNA and KI67 mRNA in 500nM doxorubicin (1day) treated MCF7 cells compared to untreated cells. H. for METTL3 mRNA (below) in Sal003 (10uM 1 day) treated MCF7 cells compared to untreated cells. I. Nascent amino acid labeling and METTL3 immunoprecipitation (Western blot of METTL3 with dark exposure of 1/10 Inputs) from 500nM doxorubicin (1day) treated versus untreated MCF7 cells to detect translation levels (quantitation below) of newly labeled immunoprecipitated METTL3 in these cells. Tubulin and Calnexin serve as Western blot loading controls. Data are average of 3 replicates +/−SEM. See also Fig. S2.

Article Snippet: Sal003 10uM was obtained from selleckchem.

Techniques: Western Blot, Phospho-proteomics, Dot Blot, Fractionation, Labeling, Immunoprecipitation, Quantitation Assay

Journal: bioRxiv

Article Title: Regulation of RNA methylation by therapy treatment, promotes tumor survival

doi: 10.1101/2023.05.19.540602

Figure Lengend Snippet: Western analyses of MCF7 cells treated with A. Thapsigargin (1uM for 1 day) to induce eIF2α phosphorylation, B. with 250nM Torin1 (2 days) to block mTOR and induce 4EBP dephosphorylation that coincides with eIF2α phosphorylation, to test METTL3 increase, as well as with mTOR activator, MHY1485 (2uM for 2 days), to inhibit mTOR and reduce 4EBP dephosphorylation and eIF2α phosphorylation, C. with 20uM Palomid (1 day) as another drug to block mTORC1 and mTORC2, and D. with 250nM BEZ235 (2 days) to dually block PI3K and mTOR, and induce 4EBP dephosphorylation and eIF2α phosphorylation. E. m6A dot blot analysis with 250nM Torin 1 (2 days) treatment of tumorspheres of tRNA (200nt cutoff microspin column purification) and ribosomal RNA depleted (ribo-zero purification) RNA from hormone-positive breast cancer patient sample BT30, where METTL3 and METTL14 increase as shown by Western blot analysis. F. 2D-TLC analysis of ribosomal RNA depleted, poly(A)-selected RNA from 10uM Sal003-treated MCF7 cells compared to buffer-treated cells, with nucleosides A and m6A marked. G. Y10B immunoprecipitation from 500nM doxorubicin (1day) treated cells to verify mRNAs with polysome association, followed by qPCR analysis of METTL3 and METTL14 mRNA and control 5.8S rRNA. Y10B immunoprecipitation from torin 1 (250nM for 2 days) treated MCF7 cells to verify mRNAs with polysome association, followed by qPCR analysis of METTL3 and METTL14 mRNA and control 5.8S rRNA. H. Nascent amino acid labeling and immunoprecipitation of METTL3, followed by Western analysis of nascently-translated and thus labeled METTL3 with Streptavidin-HRP and METTL3 antibodies to verify immunoprecipitation shown in . Actin and Tubulin are loading controls for Western blots. Data are average of 3 replicates +/−SEM. See also .

Article Snippet: Sal003 10uM was obtained from selleckchem.

Techniques: Western Blot, Phospho-proteomics, Blocking Assay, De-Phosphorylation Assay, Dot Blot, Purification, Immunoprecipitation, Control, Labeling

Journal: bioRxiv

Article Title: Regulation of RNA methylation by therapy treatment, promotes tumor survival

doi: 10.1101/2023.05.19.540602

Figure Lengend Snippet: A. To test whether the reversal of chemosurvival by Trazodone (5uM) in , was due to METTL3, doxorubicin (500nM) treated control shRNA MCF7 cells were compared with shMETTL3 cells (percentage chemosurviving cells) after treatment with buffer or Trazodone to override the eIF2α phosphorylation pathway and block METTL3-induced chemosurvival. Treatment of shMETTL3 cells with doxorubicin and with Trazodone that overrides eIF2α phosphorylation, does not cause additional loss of chemosensitivity, indicating that METTL3 and the eIF2α phosphorylation pathway targeted by this inhibitor, are in the same pathway and that the reduced chemosurvival with Trazodone in is due in part to reduced METTL3. B-C . Western blot analysis of PARP, ADAR1, phospho-eIF2α, and METTL3 with 500nM doxorubicin or 10uM Sal003 treatment for 18hrs, compared to control buffer. D. i. Immune cell migration assay with 500nM doxorubicin treated MCF7 cells and with cells co-cultured with 1uM ISRIB for 18hrs. ii. Bar graph shows more immune cell migration upon METTL3 depletion. Data are average of 3 replicates +/−SEM. Actin and Tubulin are loading controls for Western blots. See also , Tables S3-5.

Article Snippet: Sal003 10uM was obtained from selleckchem.

Techniques: Control, shRNA, Phospho-proteomics, Blocking Assay, Western Blot, Cell Migration Assay, Cell Culture, Migration

Journal: PloS one

Article Title: Upregulation of the coagulation factor VII gene during glucose deprivation is mediated by activating transcription factor 4.

doi: 10.1371/journal.pone.0040994

Figure Lengend Snippet: Figure 3. Phosphorylation of eIF2a during glucose deprivation. Western blots of extracts from HepG2 cells incubated for 6 hr in media having either 0 mM or 5 mM glucose, and without (-) or with (+) 10 mM Sal003 as shown above lanes. Antibodies against phospho-eIF2a (eIF2a,P), total eIF2a and GAPDH as a loading control, were used as indicated to the right of each blot. doi:10.1371/journal.pone.0040994.g003 Figure 4. Glucose concentration affects secreted FVII antigen levels. HepG2 cells were cultured for 24 hr in media supplemented with 10% or 1% fetal bovine serum and either high (25 mM), standard (5 mM), or low/no (1 mM or 0 mM) glucose. The concentration of secreted FVII antigen, expressed as ng per ml, was determined by ELISA. For each experimental set, the average amount secreted by cells in 25 mM glucose was considered 100%, and amounts secreted at the lower glucose concentrations were expressed as percentages +/2 SD relative to that. The number of replicates assayed at each condition is shown below the bars. Reducing the concentration of glucose significantly increased the amount of FVII secreted for each experi- mental set (p,0.001). doi:10.1371/journal.pone.0040994.g004

Article Snippet: Thapsigargin (Calbiochem EMD Millipore, Billerica, MA) at 500 nM final and Sal003 (Santa Cruz Biotechnology, Santa Cruz, CA) at 10 mM final were added to cultures for 6 hr where indicated.

Techniques: Phospho-proteomics, Western Blot, Incubation, Control, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: HDAC pharmacological inhibition promotes cell death through the eIF2α kinases PKR and GCN2

doi:

Figure Lengend Snippet: ( A ) eIF2α S/S and eIF2α A/A MEFs were treated with DMSO (con) or 10 μM vorinostat (SAHA) for 48h and 72h and were subjected to FACS analysis after propidium iodide staining. Cell death is represented by the percentage (%) of cells in SubG 1 . Histograms represent the mean cell death from three independent experiments for 48h of treatment (N=3, treated minus untreated). Bars denote S.E.M.. Statistical significance of the difference as calculated by Student's t-test is with *P<0.02. ( B ) The indicated MEFs were treated with DMSO (con) or 10 μM vorinostat for the indicated time periods. Protein extracts (50 μg) were subjected to western blot analysis for cleaved caspase 3 ( panel a) and actin ( panel b). A representative blot is shown. ( C ) HepG2 cells were treated with DMSO (con), 20 μΜ sal003, 10 μM vorinostat (SAHA) or both drugs for 24h and subjected to FACS analysis after propidium iodide staining. Cell death is represented by the percentage (%) of cells in SubG 1 . Histograms represent the mean cell death from three independent experiments for 24h of treatment (N=3). Bars denote S.E.M.. Statistical significance of the group difference as calculated by ANOVA is with *P<0.0001.

Article Snippet: Cells were treated with 10 μM of vorinostat (ChemieTek, Indianapolis, IN, USA) dissolved in DMSO, 20 μΜ of sal003 (ChemBridge, San Diego, CA, USA) dissolved in DMSO, 1 μM trichostatin A (Sigma, Oakville, ON, Canada) dissolved in DMSO or 50 mM nicotinamide (Sigma) dissolved in H 2 O.

Techniques: Staining, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting proprotein convertase subtilisin/kexin type 9 (PCSK9): from bench to bedside

doi: 10.1038/s41392-023-01690-3

Figure Lengend Snippet: Current pharmaceutical strategies to target PCSK9

Article Snippet: SAL003 , ChiCTR2000031373, Phase 1, ongoing , Shenzhen Salubris Pharmaceuticals.

Techniques: Blocking Assay, Activity Assay, Injection, Clinical Proteomics, Vaccines, Plasmid Preparation, CRISPR, Virus