acc synthases  (Medicago)

Journal: Science Advances

Article Title: NRF2 coordinates cystine import and glutathione use to sustain ferroptosis resistance and formaldehyde tolerance in cancer

doi: 10.1126/sciadv.aea1431

Figure Lengend Snippet: ( A ) Overview of cytosolic formaldehyde (FA) catabolism. ( B ) Heatmap depicting Log10 of the 50% FA inhibitory concentration (IC 50 ) in the presence or absence of l -buthionine-sulfoximine (BSO) at concentrations of 25 μM (H2170 cells), 50 μM (Jurkat, Nalm6, K562, and LUDLU-1 cells) or 100 μM (HCT116, H1299, A549, and T98G cells). ( C ) Representative FA viability curves normalized (norm.) to 0 μM FA for H1299 and A549 cells [mean ± SEM, n = 5 (H1299), n = 6 (A549), and two-way ANOVA (Tukey test between untreated (UT) and 100 μM BSO); for H1299: ** P = 0.0016, **** P < 0.0001, and ** P = 0.0023]. ( D ) Glutathione (GSH) levels upon 24-hour exposure to BSO [same concentrations as (B)] (mean ± SEM, n represented as dots, t test comparison between UT and BSO; * P = 0.0142, ** P = 0.0073, **** P < 0.0001). ( E ) Colony survival assay (CSA) in HCT116, H1299, and A549 in the presence of FA and/or 3.12 μM BSO for H1299 cells and 6.25 μM BSO for both HCT116 and A549 cells [mean ± SEM, n represented as dots, one-way ANOVA (Tukey test); for HCT116: **** P < 0.0001; for H1299: * P = 0.0106 and ** P = 0.0033]. ( F ) Left: GSH quantification in A549 cells expressing a nontargeting-control sgRNA (sgNTC) or sgRNAs against GSS (sg GSS ). Right: Viability in response to FA [mean ± SEM, n = 3]. ( G ) Immunoblots in A549 cells carrying an empty vector (pEV) or a vector coding for wild-type KEAP1 (p KEAP1 ). ( H ) Viability curves for A549/pEV or A549/pKEAP1 cells exposed to different concentrations of FA in the presence of 100 μM BSO [mean ± SEM, n = 3 and two-way ANOVA (Tukey test between UT and 100 μM BSO); **** P < 0.0001]. ( I ) DNA-protein cross-link (DPC) accumulation in A549/pEV and A549/pKEAP1 cells exposed to 100 μM BSO for 48 hours and to the indicated concentrations of FA for 4 hours (mean ± SEM, n = 3, one-way ANOVA in each cell line corrected by Tukey test) . Created in BioRender. https://BioRender.com/tsuwhsf .

Article Snippet: Nalm6 cells and Jurkat cells were obtained from DSMZ, while K562 were sourced from ATCC.

Techniques: Concentration Assay, Comparison, Clonogenic Cell Survival Assay, Expressing, Control, Western Blot, Plasmid Preparation

Journal: Molecular Oncology

Article Title: A role for eIF5A in translational control of stress‐inducible proteins in multiple myeloma cell lines

doi: 10.1002/1878-0261.70328

Figure Lengend Snippet: Relative fitness of mock vs EIF5A knockout (KO) cells in RPMI‐8226, LP‐1, and JeKo‐1 cells. (A) Competition assay in RPMI‐8226 wild‐type (WT) vs mock or vs EIF5A KO cells under basal conditions, with doxycycline (3 days, 1 μg·mL −1 ), qVD (5 μ m ) or TG (1 n m ), N = 3. Data are presented as mean ± SEM. (B) Competition assay in LP‐1 WT vs mock or vs EIF5A KO cells under basal conditions, with doxycycline (3 days, 1 μg·mL −1 n m ) or TG (1.5 n m ), N = 4. Data are presented as mean ± SD. (C) Competition assay in JeKo‐1 WT vs mock or vs EIF5A KO cells under basal conditions, with doxycycline (3 days, 1 μg·mL −1 n m ) or TG (2.5 n m ), N = 4. Data are presented as mean ± SD.

Article Snippet: RPMI‐8226 cells (RRID:CVCL_0014; obtained from ATCC), LP‐1 (RRID:CVCL_0012; obtained from DSMZ) and JeKo‐1 (RRID:CVCL_1865; obtained from DSMZ) were cultured in IMDM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: Knock-Out, Competitive Binding Assay

Journal: Molecular Oncology

Article Title: A role for eIF5A in translational control of stress‐inducible proteins in multiple myeloma cell lines

doi: 10.1002/1878-0261.70328

Figure Lengend Snippet: Inhibiting eIF5A hypusination and unfolded protein response (UPR) activity. (A) Western blot of eIF5A hypusination in RPMI‐8226 and LP‐1 cells treated with GC7 (6 μ m ), N = 3, one representative experiment is shown. (B) Quantification of A. Data are presented as mean ± SEM. (C) Expression of key UPR proteins upon in RPMI‐8226 and LP‐1 cells treated with GC7 (6 μ m ), N = 2, one representative experiment is shown. (D) Viability of RPMI‐8226 cells treated with TG (6 n m ), GC7 (6 μ m ), and qVD (5 μ m ) for 24 h. Data are presented as mean ± SEM, N = 3. (E) Viability of LP‐1 cells treated with TG (6 n m ), GC7 (6 μ m ), and qVD (5 μ m ) for 48 h. Data are presented as mean ± SEM, N = 3. (F) Western blot of eIF5A hypusination in RPMI8226 mock cells after TG treatment (24 h, 6 n m ), N = 4, one representative experiment is shown. Data are presented as mean ± SEM. (G) Western blot of eIF5A hypusination in RPMI8226 cells treated with BO (24 h, 20 n m ), N = 3, one representative experiment is shown. Data are presented as mean ± SEM. (H) Western blot of eIF5A hypusination in LP‐1 mock cells after TG treatment (24 h, 12 n m ), N = 4, one representative experiment is shown. Data are presented as mean ± SEM. P ‐values were calculated using student's t ‐tests, * P < 0.05, ** P < 0.01. TG, thapsigargin.

Article Snippet: RPMI‐8226 cells (RRID:CVCL_0014; obtained from ATCC), LP‐1 (RRID:CVCL_0012; obtained from DSMZ) and JeKo‐1 (RRID:CVCL_1865; obtained from DSMZ) were cultured in IMDM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: Activity Assay, Western Blot, Expressing

Journal: Molecular Oncology

Article Title: A role for eIF5A in translational control of stress‐inducible proteins in multiple myeloma cell lines

doi: 10.1002/1878-0261.70328

Figure Lengend Snippet: Relative fitness of mock vs EIF5A knockout (KO) cells in RPMI‐8226, LP‐1, and JeKo‐1 cells. (A) Competition assay in RPMI‐8226 wild‐type (WT) vs mock or vs EIF5A KO cells under basal conditions, with doxycycline (3 days, 1 μg·mL −1 ), qVD (5 μ m ) or TG (1 n m ), N = 3. Data are presented as mean ± SEM. (B) Competition assay in LP‐1 WT vs mock or vs EIF5A KO cells under basal conditions, with doxycycline (3 days, 1 μg·mL −1 n m ) or TG (1.5 n m ), N = 4. Data are presented as mean ± SD. (C) Competition assay in JeKo‐1 WT vs mock or vs EIF5A KO cells under basal conditions, with doxycycline (3 days, 1 μg·mL −1 n m ) or TG (2.5 n m ), N = 4. Data are presented as mean ± SD.

Article Snippet: RPMI‐8226 cells (RRID:CVCL_0014; obtained from ATCC), LP‐1 (RRID:CVCL_0012; obtained from DSMZ) and JeKo‐1 (RRID:CVCL_1865; obtained from DSMZ) were cultured in IMDM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: Knock-Out, Competitive Binding Assay