Journal: Antimicrobial Agents and Chemotherapy

Article Title: Quinacrine Inhibits Candida albicans Growth and Filamentation at Neutral pH

doi: 10.1128/AAC.03083-14

Figure Lengend Snippet: Effect of various QNC concentrations on C. albicans SC5314 filamentation on solid medium and 1,024 μg/ml QNC in liquid medium. (A) Growth of SC5314 colonies on YPD plus 10% fetal calf serum (FCS) solid medium, M199, and RPMI 1640, all with various

Article Snippet: For the biofilm studies, a standardized cell suspension was made in RPMI 1640 (Mediatech) supplemented with l -glutamine (Gibco) and buffered with 165 mM morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich) to pH 7.0, to attain a cell density of 1 × 106 cells/ml.

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Quinacrine Inhibits Candida albicans Growth and Filamentation at Neutral pH

doi: 10.1128/AAC.03083-14

Figure Lengend Snippet: Effect of 1,024 μg/ml QNC on C. albicans SC5314 biofilm formation in pH-buffered RPMI 1640. The effects of various concentrations of QNC against the formation of C. albicans SC5314 biofilms were assessed in a 96-well static microplate model. The

Article Snippet: For the biofilm studies, a standardized cell suspension was made in RPMI 1640 (Mediatech) supplemented with l -glutamine (Gibco) and buffered with 165 mM morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich) to pH 7.0, to attain a cell density of 1 × 106 cells/ml.

Techniques:

Journal: FEMS yeast research

Article Title: Secretion and filamentation are mediated by the Candida albicans t-SNAREs Sso2p and Sec9p

doi: 10.1111/1567-1364.12165

Figure Lengend Snippet: Staining the cell wall of the tetR-SSO2 hyphae The tetR-SSO2 strain was grown in buffered RPMI-1640, in the absence (−DOX) and presence (+DOX) of doxycycline for 5 hours. Following incubation, the cells were stained with calcofluor white (0.5 μg μl −1 ) and subsequently imaged under fluorescence with a DAPI filter. Calcofluor white predominantly stains chitin in the fungal cell wall, a component that confers rigidity to the cell. The white arrow highlights the absence of chitin from the growing hyphal tip of the tetR-SSO2 strain grown under de-repressing conditions.

Article Snippet: Filamentation was assayed at 37 °C in liquid RPMI-1640 supplemented with L-glutamine (Cellgro, Manassas, VA) and buffered with 165 mM MOPS (Sigma, St. Louis, MO) to pH 7.0 (referred to as “buffered RPMI-1640” throughout the manuscript).

Techniques: Staining, Incubation, Fluorescence

Journal: FEMS yeast research

Article Title: Secretion and filamentation are mediated by the Candida albicans t-SNAREs Sso2p and Sec9p

doi: 10.1111/1567-1364.12165

Figure Lengend Snippet: Filamentation of tetR-SSO2 and tetR-SEC9 in liquid medium Cells at a concentration of 5×10 6 cells ml −1 were grown in buffered RPMI-1640 at 37 °C with shaking at 200rpm. Filamentation was followed at hourly intervals by DIC microscopy. Representative DIC images using a 63X oil objective, taken at T=5 hr, are shown. Formation of the bulbous tip in the tetR-SSO2 strain grown in the presence of DOX is typical (arrow). Scale bar shown represents 5 μm.

Article Snippet: Filamentation was assayed at 37 °C in liquid RPMI-1640 supplemented with L-glutamine (Cellgro, Manassas, VA) and buffered with 165 mM MOPS (Sigma, St. Louis, MO) to pH 7.0 (referred to as “buffered RPMI-1640” throughout the manuscript).

Techniques: Concentration Assay, Microscopy

Journal: FEMS yeast research

Article Title: Secretion and filamentation are mediated by the Candida albicans t-SNAREs Sso2p and Sec9p

doi: 10.1111/1567-1364.12165

Figure Lengend Snippet: Effect of loss-of-function of SSO2 or SEC9 on the C. albicans Spitzenkörper C. albicans MLC1 -GFP was used to visualize the C. albicans Spitzenkörper in background strains THE1-CIp10, tetR-SSO2, and tetR-SEC9. Filamentation was induced in buffered RPMI-1640, in the presence (+) or absence (−) of DOX, and the fate of the Spitzenkörper was assessed over 5 hours of incubation. Fluorescent images at the tip of the growing hyphae at the 5-hr timepoint are shown, with the corresponding DIC image. C. albicans Mlc1p-GFP localized to septin rings in the tetR-SSO2 cell grown under repressing conditions (arrow heads); the same structure stains with calcofluor white (data not shown).

Article Snippet: Filamentation was assayed at 37 °C in liquid RPMI-1640 supplemented with L-glutamine (Cellgro, Manassas, VA) and buffered with 165 mM MOPS (Sigma, St. Louis, MO) to pH 7.0 (referred to as “buffered RPMI-1640” throughout the manuscript).

Techniques: Incubation

Journal: FEMS yeast research

Article Title: Secretion and filamentation are mediated by the Candida albicans t-SNAREs Sso2p and Sec9p

doi: 10.1111/1567-1364.12165

Figure Lengend Snippet: Cell morphology and cytokinesis defect in the absence of SSO2 and SEC9 Overnight cultures of strains grown in YPD were shifted to fresh media, with or without DOX. Cells were fixed and processed for ultrastructural analysis using thin-section electron microscopy. Note that images for hyphae are representative cross-sections; full length hyphae were not visualized in these TEMs due to curvature of the hyphae in and out of the z-axis. (A) Following incubation in YPD, tetR-SSO2 and tetR-SEC9 cells grown in the presence of DOX accumulate secretory vesicles (arrows) and produce wide-necked buds during cell division. Partial inward growth at the bud neck is also evident in tetR-SSO2 in the presence of DOX (double arrows). Bars represent a length of 500nm. tetR-SSO2 micrographs were imaged under a magnification of 20kX; tetR-SEC9 micrographs were imaged under a magnification of 30kX. (B) Following incubation in buffered RPMI-1640 to induce filamentation, tetR-SSO2 hyphal cells also accumulated secretory vesicles when grown in the presence of DOX (arrows), in contrast to tetR-SEC9 cells which appear like the wild-type controls (de-repressed state). Bars indicate 2 μm. All micrographs were imaged under a magnification of 12kX, except the tetR-SEC9 with DOX micrograph which was imaged under 6kX magnification.

Article Snippet: Filamentation was assayed at 37 °C in liquid RPMI-1640 supplemented with L-glutamine (Cellgro, Manassas, VA) and buffered with 165 mM MOPS (Sigma, St. Louis, MO) to pH 7.0 (referred to as “buffered RPMI-1640” throughout the manuscript).

Techniques: Electron Microscopy, Incubation

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

doi: 10.1152/ajpendo.00430.2013

Figure Lengend Snippet: Inhibition of IL-1β activity reverses macrophage-induced cytokine release by human adipocytes. Differentiated adipocytes were incubated with RPMI 1640 (control), MC medium, MC medium neutralized by IL-1β antibody (2 μg/ml), mouse IgG (2 μg/ml, as negative control), or MC medium alone (without cells) for 24 h. The release of IL-6 ( A ), MCP-1 ( B ), IL-8 ( C ) and RANTES ( D ) by adipocytes was measured as protein concentrations in cell culture medium by ELISAs. Data are means ± SD ( n = 6 per group). *** P

Article Snippet: The PBMCs (peripheral blood mononuclear cells) were isolated from the buffy layer and washed once with RPMI-1640 (without FBS or l -glutamine) by centrifuging at 350 g for 10 min. Monocytes were allowed to adhere to 25-cm2 tissue culture flasks (Corning, Amsterdam, The Netherlands) for 3 days, and then nonadherent cells were removed by several washes with primary macrophage medium (RPMI-1640 without phenol red, supplemented with 10% FCS, 2 mM l -glutamine, and 20 mM HEPES).

Techniques: Inhibition, Activity Assay, Incubation, Negative Control, Cell Culture

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

doi: 10.1152/ajpendo.00430.2013

Figure Lengend Snippet: Inhibition of IL-1β activity abolishes the inhibitory effect of macrophages on protein expression of IRS-1, PI3K p85α, and GLUT4 by human adipocytes. Differentiated adipocytes were incubated with RPMI 1640 medium (control), MC medium, MC medium neutralized by IL-1β antibody (2 μg/ml), or mouse IgG (2 μg/ml, as negative control) for 24 h. Cell lysates were analyzed by Western blotting and densitometry using antibodies to IRS-1 ( A, B ), PI3K p85α ( C, D ), and GLUT4 ( E, F ). Separate groups of adipocytes were treated with various agents as above, followed by stimulation with or without insulin (167 nmol/l) for 5 min; Akt phosphorylation at Ser 473 (pAkt) was analyzed by Western blotting and densitometry. Total Akt and GAPDH were used as loading controls ( G, H ). Representative blots are shown; data are means ± SE ( n = 3per group). * P

Article Snippet: The PBMCs (peripheral blood mononuclear cells) were isolated from the buffy layer and washed once with RPMI-1640 (without FBS or l -glutamine) by centrifuging at 350 g for 10 min. Monocytes were allowed to adhere to 25-cm2 tissue culture flasks (Corning, Amsterdam, The Netherlands) for 3 days, and then nonadherent cells were removed by several washes with primary macrophage medium (RPMI-1640 without phenol red, supplemented with 10% FCS, 2 mM l -glutamine, and 20 mM HEPES).

Techniques: Inhibition, Activity Assay, Expressing, Incubation, Negative Control, Western Blot

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

doi: 10.1152/ajpendo.00430.2013

Figure Lengend Snippet: Inhibition of IL-1β production by THP-1 macrophages reduces the effect of MC medium on protein expression of IRS-1, PI3K p85α, and GLUT4 and cytokine release by adipocytes. THP-1 macrophages were incubated with RPMI 1640 (control) or caspase-1 inhibitor (50 μM) for 48 h (with freshly changed medium at 24 h), and the culture medium was collected. Differentiated adipocytes were then treated with RPMI 1640 (control), MC medium, or MC medium in the presence of caspase-1 inhibitor for 24 h. Cell lysates were analyzed by Western blotting and densitometry, using antibodies to IRS-1, PI3K p85α, and GLUT4 (A–D ). Data are expressed as means ± SE ( n = 6 per group). Protein release of IL-6 ( E ), IL-8 ( F ), and RANTES ( G ) by adipocytes was measured by ELISAs. Data are expressed as means ± SD ( n = 6 per group). * P

Article Snippet: The PBMCs (peripheral blood mononuclear cells) were isolated from the buffy layer and washed once with RPMI-1640 (without FBS or l -glutamine) by centrifuging at 350 g for 10 min. Monocytes were allowed to adhere to 25-cm2 tissue culture flasks (Corning, Amsterdam, The Netherlands) for 3 days, and then nonadherent cells were removed by several washes with primary macrophage medium (RPMI-1640 without phenol red, supplemented with 10% FCS, 2 mM l -glutamine, and 20 mM HEPES).

Techniques: Inhibition, Expressing, Incubation, Western Blot

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

doi: 10.1152/ajpendo.00430.2013

Figure Lengend Snippet: MC medium generated from human PBMC-derived macrophages decreases protein expression of insulin signaling molecules and induces cytokine release by human adipocytes and the effect of blocking IL-1β. Differentiated human adipocytes were treated with RPMI 1640 (control), MC medium, MC medium neutralized by IL-1β antibody (15 μg/ml), MC medium neutralized by IL-1β antibody (7.5 μg/ml), and TNFα (7.5 μg/ml), mouse IgG (15 μg/ml), or IL-1RA (1 μg/ml) for 24 h. Cell lysates were analyzed by Western blotting and densitometry, using antibodies to IRS-1 ( A, B ) and GLUT4 ( C , D ). For measuring basal and insulin-stimulated Akt phosphorylation, adipocytes were incubated with RPMI 1640 or MC medium for 24 h before being stimulated with insulin (167 nmol/l) for 5 min; Akt phosphorylation at Ser 473 (pAkt) was analyzed by Western blotting and densitometry; GAPDH was used as loading controls ( E, F ). Representative blots are shown. Data are means ± SE ( n = 3 per group). * P

Article Snippet: The PBMCs (peripheral blood mononuclear cells) were isolated from the buffy layer and washed once with RPMI-1640 (without FBS or l -glutamine) by centrifuging at 350 g for 10 min. Monocytes were allowed to adhere to 25-cm2 tissue culture flasks (Corning, Amsterdam, The Netherlands) for 3 days, and then nonadherent cells were removed by several washes with primary macrophage medium (RPMI-1640 without phenol red, supplemented with 10% FCS, 2 mM l -glutamine, and 20 mM HEPES).

Techniques: Generated, Derivative Assay, Expressing, Blocking Assay, Western Blot, Incubation

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

doi: 10.1152/ajpendo.00430.2013

Figure Lengend Snippet: IL-1β mediates macrophage-induced alteration of glucose and lipid metabolism in human adipocytes. Adipocytes were treated with RPMI 1640 (control), THP-1 MC medium, MC medium neutralized by IL-1β antibody (2 μg/ml), MC medium with IgG (2 μg/ml), RPMI 1640 only (control), or RPMI 1640 with IL-1β (2 ng/ml) for 24 h. Glucose consumption was measured as the glucose concentration in culture medium by glucose oxidase method ( A ). Lipolysis was determined as glycerol release into culture medium ( B, C ). Data are means ± SE ( n = 6 per group). ** P

Article Snippet: The PBMCs (peripheral blood mononuclear cells) were isolated from the buffy layer and washed once with RPMI-1640 (without FBS or l -glutamine) by centrifuging at 350 g for 10 min. Monocytes were allowed to adhere to 25-cm2 tissue culture flasks (Corning, Amsterdam, The Netherlands) for 3 days, and then nonadherent cells were removed by several washes with primary macrophage medium (RPMI-1640 without phenol red, supplemented with 10% FCS, 2 mM l -glutamine, and 20 mM HEPES).

Techniques: Concentration Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

doi: 10.1152/ajpendo.00430.2013

Figure Lengend Snippet: Macrophage-conditioned (MC) medium reduces protein expression of insulin signaling molecules in human adipocytes. Adipocytes (at day 12 postdifferentiation) were treated with RPMI 1640 medium (control) or THP-1 MC medium (25%) for 24 h. Cell lysates were analyzed by Western blotting and densitometry, using antibodies to IRS-1 and PI3K p85α ( A, B, C ) and GLUT4 ( D, E ). For measuring basal and insulin-stimulated Akt phosphorylation, adipocytes were incubated with RPMI 1640 or MC medium for 24 h before being stimulated with insulin (167 nmol/l) for 5 min; Akt phosphorylation at Ser 473 (pAkt) was analyzed by Western blotting and densitometry. Total Akt and GAPDH were used as loading controls ( F, G ). Representative blots are shown; data are means ± SE ( n = 3 per group). * P

Article Snippet: The PBMCs (peripheral blood mononuclear cells) were isolated from the buffy layer and washed once with RPMI-1640 (without FBS or l -glutamine) by centrifuging at 350 g for 10 min. Monocytes were allowed to adhere to 25-cm2 tissue culture flasks (Corning, Amsterdam, The Netherlands) for 3 days, and then nonadherent cells were removed by several washes with primary macrophage medium (RPMI-1640 without phenol red, supplemented with 10% FCS, 2 mM l -glutamine, and 20 mM HEPES).

Techniques: Expressing, Western Blot, Incubation

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

doi: 10.1152/ajpendo.00430.2013

Figure Lengend Snippet: Inhibition of IL-1β activity reduces the effect of macrophages on expression profile of genes related to glucose metabolism, insulin signaling, and inflammation in human adipocytes. To block the activity of IL-1β, MC medium was preincubated with an IL-1β neutralizing antibody (2 μg/ml) for 1 h at 37°C. Differentiated adipocytes were then treated with RPMI 1640 (control), MC medium, or MC medium neutralized by IL-1β antibody for 24 h. Expression levels of genes involved in glucose metabolism, insulin signaling, lipid metabolism, insulin sensitivity ( A ), and inflammation ( B ) were determined using a PCR array. Data are expressed as fold changes ( n = 3 per group). * P

Article Snippet: The PBMCs (peripheral blood mononuclear cells) were isolated from the buffy layer and washed once with RPMI-1640 (without FBS or l -glutamine) by centrifuging at 350 g for 10 min. Monocytes were allowed to adhere to 25-cm2 tissue culture flasks (Corning, Amsterdam, The Netherlands) for 3 days, and then nonadherent cells were removed by several washes with primary macrophage medium (RPMI-1640 without phenol red, supplemented with 10% FCS, 2 mM l -glutamine, and 20 mM HEPES).

Techniques: Inhibition, Activity Assay, Expressing, Blocking Assay, Polymerase Chain Reaction

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

doi: 10.1152/ajpendo.00430.2013

Figure Lengend Snippet: Blocking IL-1 receptor with IL-1 receptor antagonist reverses the effects of MC medium on protein expression of IRS-1, PI3K p85α, and GLUT4 and cytokine release by human adipocytes. Differentiated adipocytes were incubated with RPMI 1640 (control), MC medium, or MC medium with a recombinant IL-1 receptor antagonist (IL-1RA; 1 μg/ml) for 24 h. Cell lysates were analyzed by Western blotting and densitometry, using antibodies to IRS-1 ( A, B ), PI3K p85α ( C , D ), and GLUT4 ( E, F ). On the blot shown, there is an empty lane between MC and MC+IL-1RA groups ( C ). Data are expressed as means ± SE ( n = 3 per group). * P

Article Snippet: The PBMCs (peripheral blood mononuclear cells) were isolated from the buffy layer and washed once with RPMI-1640 (without FBS or l -glutamine) by centrifuging at 350 g for 10 min. Monocytes were allowed to adhere to 25-cm2 tissue culture flasks (Corning, Amsterdam, The Netherlands) for 3 days, and then nonadherent cells were removed by several washes with primary macrophage medium (RPMI-1640 without phenol red, supplemented with 10% FCS, 2 mM l -glutamine, and 20 mM HEPES).

Techniques: Blocking Assay, Expressing, Incubation, Recombinant, Western Blot

Journal: Cell Cycle

Article Title: Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

doi: 10.4161/cc.27818

Figure Lengend Snippet: Figure 6. Cell cycle kinetics of cancer cells during seeding on Gelffoam®. FUCCI-expressing cancer cells (5 × 10 6 ) were placed on Gelfoam® (1 × 1 cm) in RPMI 1640 medium. ( A ) Low-magnification image of an overview

Article Snippet: FUCCI-expressing MKN45 cells were seeded on plastic plates for 2-dimensional culture in RPMI-1640 medium (Mediatech).

Techniques: Expressing

Journal: Cell Cycle

Article Title: Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

doi: 10.4161/cc.27818

Figure Lengend Snippet: Figure 5. Comparison of migration velocity of cancer cells in G 0 /G 1 phase and S/G 2 /M phases during cell division. FUCCI-expressing cancer cells (5 × 10 6 ) were placed on Gelfoam® (1 × 1 cm) in RPMI 1640 medium. ( A ) Cancer

Article Snippet: FUCCI-expressing MKN45 cells were seeded on plastic plates for 2-dimensional culture in RPMI-1640 medium (Mediatech).

Techniques: Migration, Expressing

Journal: Cell Cycle

Article Title: Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

doi: 10.4161/cc.27818

Figure Lengend Snippet: Figure 7. Chemotherapy does not kill or inhibit the movement of single invading cancer cells in G 0 /G 1 phase. FUCCI-expressing cancer cells (2 × 10 6 ) were placed on Gelfoam® (1 × 1 cm) in RPMI 1640 medium. Three days after

Article Snippet: FUCCI-expressing MKN45 cells were seeded on plastic plates for 2-dimensional culture in RPMI-1640 medium (Mediatech).

Techniques: Expressing

Journal: Cell Cycle

Article Title: Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

doi: 10.4161/cc.27818

Figure Lengend Snippet: Figure 4. Behavior of individual FUCCI-expressing cancer cells cultured on Gelfoam®. FUCCI-expressing cancer cells (5 × 10 6 ) were placed on Gelfoam® (1 × 1 cm) in RPMI 1640 medium. ( A ) Low-magnification image of

Article Snippet: FUCCI-expressing MKN45 cells were seeded on plastic plates for 2-dimensional culture in RPMI-1640 medium (Mediatech).

Techniques: Expressing, Cell Culture

Journal: Cell Cycle

Article Title: Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

doi: 10.4161/cc.27818

Figure Lengend Snippet: Figure 2. Cell cycle distribution of invasive and noninvasive cancer cells. FUCCI-expressing cancer cells (1 × 10 7 ) were placed on Gelfoam® (1 × 1 cm) in RPMI 1640 medium. ( A ) High-magnification real-time images of a small

Article Snippet: FUCCI-expressing MKN45 cells were seeded on plastic plates for 2-dimensional culture in RPMI-1640 medium (Mediatech).

Techniques: Expressing

Journal: Cell Cycle

Article Title: Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

doi: 10.4161/cc.27818

Figure Lengend Snippet: Figure 3. Invasive cancer cells are predominantly in G 0 /G 1 . FUCCI-expressing cancer cells (5 × 10 6 ) were placed on Gelfoam® (1 × 1 cm) in RPMI 1640 medium. ( A ) High-magnification real-time images of invading cancer cells

Article Snippet: FUCCI-expressing MKN45 cells were seeded on plastic plates for 2-dimensional culture in RPMI-1640 medium (Mediatech).

Techniques: Expressing

Journal: bioRxiv

Article Title: Methionine synthase is essential for cancer cell proliferation in physiological folate environments

doi: 10.1101/2020.06.12.149005

Figure Lengend Snippet: MTR knockout reduces SAM levels but not methionine levels. (A) Proliferation rates of A549 cells without (+EV) or with MTR expression (+MTR) cultured in the indicated folate with or without the addition of 1 mM methionine to supplement the 100 μM methionine present in RPMI-1640 culture media. (B) LC/MS measurement of intracellular methionine, lysine, and serine levels in A549 cells without (+EV) or with MTR expression (+MTR) cultured for 4 days in the indicated folate. Data are normalized to the total protein content of cells in each condition and to an internal standard. (C) LC/MS measurement of intracellular SAM and SAH levels in A549 cells without (+EV) or with MTR expression (+MTR) cultured for 4 days in the indicated folate. Data are normalized to the total protein content of cells in each condition and to an internal standard. (D) Western blots to assess levels of mono- or dimethyl lysine-containing proteins, RPS6 phosphorylated at S235/6, total RPS6, S6K phosphorylated at T389, and total S6K in A549 cells without (+EV) or with MTR expression (+MTR). Phosphorylation index is defined as the phosphorylated protein antibody signal divided by the same total protein antibody signal in each lane. (E) Proliferation rates of A549 cells without (+EV) or with MTR expression (+MTR) cultured in the indicated folate with or without the addition of 1 mM SAM. Mean +/- SD is displayed for all panels. p values indicated on all panels are derived from two-tailed, unpaired Welch’s t tests.

Article Snippet: Cells were trypsinized, counted and plated into six well dishes (Corning Life Sciences) in 2 mL of RPMI-1640 medium lacking folic acid and incubated overnight.

Techniques: Knock-Out, Expressing, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Western Blot, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Methionine synthase is essential for cancer cell proliferation in physiological folate environments

doi: 10.1101/2020.06.12.149005

Figure Lengend Snippet: MTR expression affects SAM but not methionine metabolism in T.T cells. (A) Proliferation rates of T.T cells without (+EV) or with MTR expression (+MTR) cultured in the indicated folate with or without the addition of 1 mM methionine to supplement the 100 μM methionine present in RPMI-1640 culture media. (B) LC/MS measurement of intracellular methionine, lysine, and serine levels in T.T cells without (+EV) or with MTR expression (+MTR) cultured for 4 days in the indicated folate. (C) LC/MS measurement of intracellular SAM and SAH levels in T.T cells without (+EV) or with MTR expression (+MTR) cultured for 4 days in the indicated folate. (D) Western blots to assess levels of mono- or dimethyl lysine-containing proteins, RPS6 phosphorylated at S235/6, total RPS6, S6K phosphorylated at T389, and total S6K in T.T cells without (+EV) or with MTR expression (+MTR). Phosphorylation index is defined as the phosphorylated protein antibody signal divided by the same total protein antibody signal in each lane. (E) Proliferation rates of T.T cells without (+EV) or with MTR expression (+MTR) cultured in the indicated folate with or without the addition of 1 mM SAM. Mean +/- SD is displayed for all panels. p values indicated on all panels are derived from two-tailed, unpaired Welch’s t tests. For LC/MS measurements, data are normalized to the total protein content of cells in each condition and to an internal standard.

Article Snippet: Cells were trypsinized, counted and plated into six well dishes (Corning Life Sciences) in 2 mL of RPMI-1640 medium lacking folic acid and incubated overnight.

Techniques: Expressing, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Western Blot, Derivative Assay, Two Tailed Test