Journal: Aging (Albany NY)

Article Title: Elevated ceramides 18:0 and 24:1 with aging are associated with hip fracture risk through increased bone resorption

doi: 10.18632/aging.102389

Figure Lengend Snippet: The association of plasma ceramides with bone mineral density and bone turnover marker.

Article Snippet: C18:0 and C24:1 ceramides were purchased from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Marker

Journal: Leukemia

Article Title: The dual epigenetic role of PRMT5 in acute myeloid leukemia: gene activation and repression via histone arginine methylation

doi: 10.1038/leu.2015.308

Figure Lengend Snippet: (A) Immunoprecipitation (IP) assay in FLT3-WT THP1 and FLT3-ITD MV4–11 cell lines; pulling down was performed with anti-PRMT5 and immunostaining with anti-PRMT5 and anti-FLT3. No physical association between PRMT5 and FLT3 was detected. (B) Inhibition of FLT3 kinase activity did not influence overall phosphorylated PRMT5 levels. Whole cell lysate was used to pull down phosphorylated tyrosine residues (anti-p-Tyr) in cells treated with FLT3 inhibitor. Phosphorylated ERK1/2 (p-ERK1/2) level was detected as control for p-Tyr IP and effectiveness of kinase inhibitory effects of treatment. (C) Western blotting of treatment of MV4–11 cells with PKC412, specific FLT3 inhibitor (FLT3i), and HLCL-61 for 5 hours. Levels of phosphorylated STAT5 (pSTAT5) and pERK1/2 were detected as positive control to confirm the effective FLT3 inhibition. Immunostaining was carried out sequentially after stripping the membrane following staining with anti-FLT3, anti-pFLT3, anti-Sp1, anti-PRMT5, anti-pSTAT5, anti-pERK1/2, and anti-GAPDH. (D) Treatment of AML cell lines and primary blasts with HLCL-61 led to significant suppression of FLT3 mRNA and protein levels. qRT-PCR was used to measure relative FLT3 mRNA levels and Western blotting to detect protein levels of FLT3 and GAPDH. (E-G) Small RNA interference knockdown of PRMT5 resulted in significant downregulation of FLT3 mRNA and protein expression in different AML cells. (H) Forcing PRMT5 expression in THP-1 cells resulted in FLT3 mRNA and protein upregulation.

Article Snippet: PKC412 (Sigma-Aldrich, M1323) and FLT3 inhibitor (Calbiochem #343020) were purchased, whereas HLCL-61 (HLCL-61) was prepared by Hongshan Lai at Ohio State University.

Techniques: Immunoprecipitation, Immunostaining, Inhibition, Activity Assay, Western Blot, Positive Control, Stripping Membranes, Staining, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: The transcription factor STAT5 catalyzes Mannich ligation reactions yielding inhibitors of leukemic cell proliferation

doi: 10.1038/s41467-018-07923-2

Figure Lengend Snippet: Synergy of inhibitor 16 and kinase inhibitor midostaurin (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of < 1, = 1, and > 1 indicate synergism, additivity, and antagonism, respectively. Error bars denote mean ± S.D. d Relative STAT5 phosphorylation levels were plotted based on the raw data from western blotting chemiluminescence readings to study the synergistic effect of both compounds on STAT5 phosphorylation reduction. MV-411 cells were treated with compound 16 or PKC412 alone or in combination and incubated for 6 h ( n = 3, error bars denote mean ± S.D.). e The corresponding body weight changes in non-xenografted mice during compound 16 treatments ( n = 6, error bars denote mean ± S.D.). f Compound 16 significantly inhibits tumor growth in BaF3/FLT3-ITD xenograft tumor model. Time course of tumor growth suppressed by compound 16 (200 mg kg −1 ) in mice bearing BaF3/FLT3-ITD tumor ( n = 6, error bars denote mean ± S.D.)

Article Snippet: Recombinant mouse IL3 protein (cat. PMC0034, 1 ng ml −1 ) was purchased from Thermo Fisher Scientific and midostaurin hydrate (PKC412, cat. M1323) was from Sigma-Aldrich.

Techniques: Activity Assay, Incubation, Staining, Flow Cytometry, ATP Bioluminescent Assay, Generated, Software, Inhibition, Western Blot