Histories of drug exposure of the NHL matched control groups.

<p>NSAID, non-steroidal anti-inflammatory drug; ACEI, angiotensin-converting enzyme inhibitor; COX-2, cyclooxygenase-2</p><p>Histories of drug exposure of the NHL matched control groups.</p

Demographic characteristics of the NHL group and the matched control group.

<p>*72 subjects, including 4 in the NHL group and 68 in the control group, were not stratified. HBV, hepatitis B virus; HCV, hepatitis C virus; DM, diabetes mellitus; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus</p><p>Demographic characteristics of the NHL group and the matched control group.</p

Crude and adjusted odds-ratios of non-Hodgkin lymphoma (NHL) associated with previous statin administration during the follow-up period in the study cohort.

<p>OR, odds ratio; CI, confidence interval; cDDD, cumulative defined daily dose.</p><p>*Adjusted for age, gender, urbanization level, HBV infection, HCV infection, autoimmune diseases (RA, SLE and sicca syndrome), diabetes mellitus, hypertension, hyperlipidemia, and the uses of ACEI, NSAID, COX-2 inhibitors and aspirin, fibrate and other lipid-lowering drugs.</p><p>Crude and adjusted odds-ratios of non-Hodgkin lymphoma (NHL) associated with previous statin administration during the follow-up period in the study cohort.</p

Expression of putative <i>miR-375</i> target genes in DOX-induced senescent K562 cells.

<p>(A) mRNA expression of putative <i>miR-375</i> target genes in K562 cells treated with 50 nM DOX for 3 and 4 days as measured by real-time quantitative RT-PCR. The value of the mRNA expression in untreated K562 cells of the same day is designated 1, and the level of mRNA expression of DOX-treated K562 cells are calibrated to this value. Data represented are the means and SE of 5 independent experiments. (B) Expression of <i>14-3-3zeta</i>, <i>LDHB</i>, and <i>SP1</i> genes in K562 cells treated with 50 nM DOX (<i>DOX</i>) transfected with 100 nM <i>has-</i>anti-<i>miR-375</i> inhibitor followed by 50 nM DOX treatment (<i>Inh</i>) or transfected with 100 nM <i>has</i>-<i>miR-375</i> precursor (<i>Pre</i>) for 3 and 4 days. The calculation of gene expression was as described in (A). Data represented are the means and SE of 3 independent experiments.</p

DOX induced senescence but PTX not senescence in K562 cells.

<p>(A) K562 cells treated with 50 nM of DOX for 4 days were stained for SA-β-gal activity followed by DAPI staining. Original magnification is 400×. Representative microscopic fields are shown. (B) K562 cells were treated with 50 nM of DOX for 5 days, and the percentages of apoptotic cells were determined by Annexin V/PI staining followed by flow cytometric analysis. Data represented are the means and SE of 3 independent experiments. (C) K562 cells were treated with 50 nM of DOX for 5 days, and DNA contents were measured by flow cytometric analysis after PI staining. Data represented are the means and SE of 3 independent experiments.</p

mRNA expression of senescence-associated genes in K562 cells treated with 50 nM DOX as measured by real-time quantitative RT-PCR.

<p>The x-axis indicates the days post DOX treatment and the y-axis represents the relative mRNA expression level. The value of the mRNA expression at day 0 is designated 1, and the levels of all other days are calibrated to this value. Data represented are the means and SE of 5 independent experiments.</p

<i>miR-375</i> is upregulated in DOX-induced senescent K562 cells.

<p>(A) miRNAs upregulated in 50 nM DOX-treated K562 cells for 4 days as measured by TaqMan® microRNA microarray analysis. The value of the miRNA expression in untreated K562 cells of day 4 is designated 1, and the level of miRNA expression of DOX-treated K562 cells are calibrated to this value. Data represented are the means and SE of 3 independent experiments. (B) Validation of miRNA expression by individual mature TaqMan® microRNA assays using real-time quantitative RT-PCR. The 4 most strongly expressed miRNAs selected from TaqMan® microRNA microarray analysis were further validated. The value of the miRNA expression in untreated K562 cells is designated 1, and the level of miRNA expression of DOX-treated K562 cells of the same day are calibrated to this value. Data represented are the means and SE of 3 independent experiments. (C) Inhibition of <i>has</i>-<i>miR-375</i> by 100 nM <i>has</i>-anti-<i>miR-375</i> inhibitor or 100 nM <i>has</i>-anti-<i>miR-375</i> inhibitor scramble negative control (SC) in K562 cells. After transfection for 48 hours, K562 cells were treated with 50 nM DOX for 5 days. The expression of mature <i>has</i>-<i>miR-375</i> was examined by TaqMan® microRNA assays using real-time quantitative RT-PCR. The value of the <i>has</i>-<i>miR-375</i> expression at day 0 is designated 1, and the levels of all other days of the same treatment are calibrated to this value. Data represented are the means and SE of 5 independent experiments. (D) WST-1 assay was performed to determine cell proliferation after 100 nM <i>has-</i>anti-<i>miR-375</i> inhibitor or 100 nM <i>has</i>-anti-<i>miR-375</i> inhibitor SC transfection followed by 50 nM DOX treatment in K562 cells. Data represented are the means and SE of 5 independent experiments. *Indicates significant difference compared to cells treated with 50 nM DOX and treated with 100 nM anti<i>-miR-375</i> SC and 50 nM DOX (<i>p</i><0.05). (E) Overexpression of <i>miR-375</i> by 100 nM <i>has</i>-<i>miR-375</i> precursor or 100 nM <i>has</i>-<i>miR-375</i> precursor SC in K562 cells. The measurement and calculation of mature <i>has</i>-<i>miR-375</i> expression were as described in (C). Data represented are the means and SE of 5 independent experiments. (F) WST-1 assay was performed to determine cell proliferation after 100 nM <i>has</i>-<i>miR-375</i> precursor or 100 nM <i>has</i>-<i>miR-375</i> precursor SC transfection in K562 cells. Data represented are the means and SE of 5 independent experiments. *Indicates significant difference compared to both untreated K562 cells and cells treated with 100 nM <i>has</i>-<i>miR-375</i> precursor SC (<i>p</i><0.05).</p

Additional file 1 of Indigofera suffruticosa aerial parts extract induce G2/M arrest and ATR/CHK1 pathway in Jurkat cells

Additional file 1: Supplementary Table S1. Antibodies used in this study. Supplementary Fig. S1. Representative plot of Annexin V staining results. This figure is related to Fig. 2D. Supplementary Fig. S2. The caspase-3/7 activities in Jurkat cells after 12 h of ISAE treatment were assessed by Caspase-Glo® 3/7 assay (Promega) according to the manufacturer's instructions. The graph was expressed as fold changes to control group. Supplementary Fig. S3. Relative quantitative analysis of tryptanthrin, indigo, and indirubin in ISAE. The selected ion current chromatograms of the ISAE extract, tryptanthrin, indigo, and indirubin were carried out through MS full scan experiment in positive mode. The relative abundance of the three selected compounds in ISAE extract were calculated based on comparing the peak area ratios with standard compounds. Supplementary Fig. S4. Cell gating for cell cycle analysis. For cell cycle analysis, cells were gated using forward scatter (FSC) and side scatter (SSC) properties. This helped in excluding cell debris and selecting the desired cells (within the black circle) for analysis. The gating area was established based on the solvent control group (0 μg/mL of ISAE) and was consistently applied to all other groups. This illustration corresponds to Fig. 2A. Original images of western blot. Multiple exposure images of WB

Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

<div><p>We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8⁺CD44^high) T cells displaying a CD25⁻NKG2D⁺ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.</p></div

Anti-tumor effects of IT in CD4 knockout mice.

<p>3LL tumor bearing WT or CD4 knockout (B6.129S2-CD4^tm1Mak/J) mice were treated with IT or PBS/rIgG (control) and survival and tumor growth were measured. For <i>in vivo</i> tumor studies one million 3LL cells were administered by s.c. injection into the flank of C57BL/6 mice seven days prior to initiation of therapy. (<b>a</b>) Survival. (<b>b</b>) Mean tumor volume with SEM. (<b>c–f</b>) Growth plots of individual tumors in each group. N = 12 mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p