DNP induces motility and invasion through HSP70-2.

<p>A, 6-10B cells were transfected using pcDNA3.1-HSP70-2, pcDNA3.1, pU6pro-si-HSP70-2, or pU6pro-si-mock, and then the stable cell lines 6-10B-HSP70-2, 6-10B -si-HSP70-2, 6-10B-pcDNA3.1, and 6-10B-si-mock were obtained by selection for G418 resistance. HSP70-2HSP70-2 expression was detected in these cells with or without DNP treatment. β-actin is shown as a loading control. The motility (B) and invasion (C) of 6-10B - HSP70-2, 6-10B-si-HSP70-2, 6-10B-pcDNA3.1, and 6-10B-si-mock with or without DNP treatment were detected. Data are presented as means ± SD from 3 independent experiments. Results were analyzed by One-way ANOVA with post-hoc Dunnett’s test (*, p<0.05).</p

Dose- and time- dependent DNP-mediated HSP70 expression.

<p>A, HSP70-2 expression was detected in NPC cell lines 5-8F, 6-10B, CNE1, CNE2, HONE1, HNE1, and HNE2 by Western blotting. B, DNP-mediated HSP70-2 expression was detected using immunofluorescence. a, 6-10B cells treated with 0.1% DMSO-PBS served as a negative control; b, 6-10B cells treated with DNP; c, 5-8F served as a positive control; d, 6-10B cells stained with DAPI. Scale bar = 50 µm. C, DNP-mediated HSP70-2 expression was detected in 6-10B and CNE2 cells. D, Time- and dose-dependency of DNP-induced HSP70-2 expression. a, HSP70-2 expression in 6-10B cells treated with the indicated concentration; b, HSP70-2 expression in 6-10B cells with 4 µM DNP for the indicated time. Three independent experiments were performed, the abundance ratio to β-actin was counted, and data are represented as means ± SD from three experiments. *, p<0.05. E, Expression of HSP70-2, HSP70t, HSC70, GRP75, GRP78, and HSP70-4 was detected in 6-10B cells after DNP treatment.</p

Non-cytotoxic concentration of <i>N</i>,<i>N</i>′-dinitrosopiperazine (DNP) in 6-10B cells.

<p>A, Structure of DNP, an <i>N</i>-nitroso compound. B, 6-10B cells were treated with the indicated concentration DNP for 48 h before being subjected to the MTT cell viability assay. C, After 6-10B cells were treated with the indicated DNP concentration for 48 h, cell media were subjected to the LDH assay. The optical density (OD) indicates the relative OD at 563 nm. Lactate dehydrogenase (LDH) activity is expressed per 1 L of media. Data are presented as means ± SD from 3 independent experiments. Statistical analysis was performed using the Student’s <i>t</i> test (*, <i>p</i><0.05).</p

DNP-mediated HSP70 RNA transcription through HSP70-2 promoter interaction.

<p>A, DNP-mediated Hsp70 transcription was detected in 6-10B and CNE1 cells by RT-PCR. B, Schematic of the HSP70-2 promoter and its mutations. HSP70-2 promoter, the full length of promoter; HSP70-2M1, HER1 promoter mutation; HSP70-2M2, HER1, and HER2 mutation; HSP70-2M3, HER1, 2 and HSE mutation. HSP70-2 promoter and its mutations were respectively incubated with ³H-DNP, and the reactive complexes were separated by SDS-PAGE for autoradiography. C, The affinity was determined by displacement of ³H-DNP from the HSP70-2 promoter. IC50 of DNP interaction with HSP70-2 promoter was 273.3 nM, and Ki was 107 nM. D, Relative firefly luciferase activities were normalized by the Renilla luciferase activity (mean ± SD). The relative luciferase activity indicates the transcriptional activity of the HSP70-2 promoter and its mutation with or without DNP treatment.</p

DNP-mediated NPC cell metastasis and HSP70-2 expression <i>in vivo</i>.

<p>A, 20 BABL/c nude mice were injected with 6-10B cells in Matrigel through the tail vein (1×10⁴ cells/mouse), and randomly divided into 2 groups (DNP-treated and control groups) with 10 mice per group. The DNP-treated group was abdominally injected using DNP at a dose of 40 mg/kg twice a week for 60 days. The control group was injected using 0.1% DMSO-PBS. After 60 days, metastatic tumors from the mediastinal lymph nodes were weighed (*, p<0.05). B, HSP70-2 expression was detected in the metastatic tumor samples using immunohistochemistry. Paraffin sections were stained using hematoxylin and eosin as well as with antibodies against HSP70-2. The upper panel represents staining with hematoxylin and eosin; the lower panel represents immunohistochemistry data. a and c represent untreated 6-10B cells as a negative control; b and d represent DNP-treated 6-10B cells; Arrows = positive cell. Original magnification, ×400. Scale bar = 5 µm. C, Schematic illustration of DNP-induced HSP70-2. DNP-mediated HSP70-2 expression through binding to HSP70-2 promoter, promotes motility and invasion, leading to metastasis of NPC cells.</p

DNP-mediated NPC cell metastasis <i>in vitro</i>.

<p>A, Matrigel-coated Boyden chambers was used to measure 6-10B cell invasion, and an uncoated Boyden chamber was used to determine cell motility after DNP treatment. The cells invaded to the lower surface of the membrane were fixed with methanol and stained with hematoxylin and eosin. a and c, 6-10B cells treated with 0.1% DMSO-PBS; b and d, 6-10B cells treated with 4 µM DNP; Scale bar = 10 µm. Arrows = invaded cell. In dose-dependency assays, 6-10B cells were treated with the indicated concentrations DNP. In time-course assays, cells were treated with 4 µM DNP for the indicated time. Treated cells were subjected to motility and invasion analysis. Images were taken under a light microscope and random fields of view were counted to determine the number of invading cells. B, Invasion of 6-10B cells at various concentrations; C, Invasion of 6-10B cells at various time points; D, Motility of 6-10B cells at various concentrations; and E, Motility of 6-10B cells at various time points. Statistically analysis performed using a one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test (*, p<0.05).</p

si-AGR2 decreased DNP-mediated invasion and motility in 5–8F cells.

<p>5–8F cells were transfected with pUprosi-AGR2 (si-AGR2) or pUprosi-mock (si-mock), and 5–8F-si-AGR2 and 5–8F-si-mock were obtained by selection for G418 resistance, and treated with DNP. A, AGR2 expression was detected using western blotting. A Boyden chamber was used to measure the invasion and motility of 5–8F-si-AGR2 and 5–8F-si-mock with or without DNP treatment. The cells that invaded the membrane were fixed with methanol and stained with hematoxylin and eosin. Random fields of view were counted to determine the number of invading cells. B, invasion of the treated cells. C, motility of the treated cells. The number of traversed cells was counted in three individual experiments and presented as the mean ± SD. *indicates P<0.05.</p

DataSheet_1_Association between immunoglobulin G N-glycosylation and lupus nephritis in female patients with systemic lupus erythematosus: a case-control study.docx

BackgroundLupus nephritis (LN) is a crucial complication of systemic lupus erythematosus (SLE) and has important clinical implications in guiding treatment. N-glycosylation of immunoglobulin G (IgG) plays a key role in the development of SLE by affecting the balance of anti-inflammatory and proinflammatory responses. This study aimed to evaluate the performance of IgG N-glycosylation for diagnosing LN in a sample of female SLE patients.MethodsThis case-control study recruited 188 women with SLE, including 94 patients with LN and 94 age-matched patients without LN. The profiles of plasma IgG N-glycans were detected by hydrophilic interaction chromatography with ultra-performance liquid chromatography (HILIC-UPLC). A multivariate logistic regression model was used to explore the associations between IgG N-glycans and LN. A diagnostic model was developed using the significant glycans as well as demographic factors. The performance of IgG N-glycans in the diagnosis of LN was evaluated by receiver operating characteristic (ROC) curve analysis, and the area under the curve (AUC) and its 95% confidence interval (CI) were calculated.ResultsThere were significant differences in 9 initial glycans (GP2, GP4, GP6, GP8, GP10, GP14, GP16, GP18 and GP23) between women with SLE with and without LN (P ConclusionOur findings indicate that decreased sialylation, galactosylation, and core fucosylation and increased bisecting GlcNAc might play a role in the development of LN by upregulating the proinflammatory response of IgG. IgG N-glycans can serve as potential biomarkers to differentiate individuals with LN among SLE patients.</p

Immunohistochemical staining of AGR2, CTSB, and CTSD expressions in normal nasopharyngeal epithelial tissues (NNET), nasopharyngeal carcinoma (NPC), and cervical lymph node metastatic NPC (LMNPC) tissues.

<p>The formalin-fixed and paraffin-embedded NNET, NPC, and LMNPC tissue sections were stained using a standard immunohistochemical technique. a, AGR2 expression in NNET; b, AGR2 expression in NPC; c AGR2 expression in LMNPC; d, CTSB expression in NNET; e, CTSB expression in NPC; f, CTSB expression in LMNPC; g, CTSD expression in NNET; h, CTSD expression in NPC; i, CTSD expression in LMNPC; j, NNET hematoxylin and eosin; k, NPC hematoxylin and eosin; l, LMNPC hematoxylin and eosin. Arrows, positive cells. Original magnification, ×400. Scale bar, 5 μm.</p

Correlation of AGR2 with CTSB and CTSD expression in NPC and LMNPC tissues.

<p>Note: <i>r</i> represents correlation coefficient.</p