Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanistic insights into CpG DNA and IL-15 synergy in promoting B-cell chronic lymphocytic leukemia clonal expansion 1

doi: 10.4049/jimmunol.1800591

Figure Lengend Snippet: A. B-CLL clones (U-791, U-1239, U-1953) were cultured for varying intervals (3h, 9h, and 20h) with medium alone or with ODN, prior to RNA isolation. Specific mRNA levels were determined by qRT-PCR performed as described in Materials & Methods, using GAPDH housekeeping control mRNA to calculate ∆Ct values. ∆Ct values within medium versus ODN treated B-CLL cultures for IL15RA (left plot) and IL2RB (CD122) (right plot) are shown. Statistical evaluations for differences were made with paired T-tests. * indicates that ∆Ct values for specific mRNA in medium-treated cells is significantly different (P≤ 0.03) by 1-sided T-tests; ** indicates that ∆Ct values in medium versus ODN treated cells is significantly different (P≤ 0.04) by 2-sided T-tests. B. ∆Ct values were used to calculate ∆∆Ct values, using medium-treated cells as control baseline and fold-change above medium only obtained by 2^(−∆∆Ct) (31). Plotted are the fold-change values for IL15RA mRNA (left) and IL2RB (right) at increasing intervals after ODN stimulation. Represented are data from the 3 individual B-CLL clones. C. Intracellular immunofluorescent staining with APC-labeled anti-p65 NFkB(Ser529) mAb (solid line) and APC-labeled IgG control (filled grey) was used to confirm that p65/RelA is activated in ODN-2006-exposed B-CLL. A gate was placed in the fluorescence histogram to indicate the threshold for positivity (set at 1% positive cells with IgG staining control). P value indicates statistical significance by 1-sided, paired T-test. E and F. NF-kB inhibitor reduces IL15RA and IL2RB (CD122) mRNA. The effect of 30 min pre-exposure to NF-kB inhibitor, BAY-11-9802 (1.25 and/or 2.5 μM) on ODN-triggered increases in specific mRNA was evaluated in two B-CLL clones: (E) M-1031 and (F) U-1239. M-1031 CLL cells were cultured for 3h with medium alone or ODN prior to mRNA isolation, with sets of parallel cultures pre-exposed for 30 min to NF-kB inhibitor or DMSO vehicle control. Specific RNA levels were determined as above and are represented as fold-change above that in medium only cultures with vehicle (shown by dotted line). U-1239 CLL cells were treated as above, but two culture periods were employed (3h and 9h) to permit optimal detection of IL2RB mRNA. * indicates that specific mRNA levels in cultures with NF-kB inhibitor are significantly different from those in parallel cultures with vehicle: P< 0.05 upon data analysis by 2-sided, unpaired T-tests.

Article Snippet: Staining was performed in duplicate with cells in PW buffer supplemented with Alexa Fluor 647-conjugated anti-phospho-p65(Ser529) mAb (BD Pharmingen), a validated mAb ( 29 ), or with similarly labeled mouse IgG2b control.

Techniques: Clone Assay, Cell Culture, Isolation, Quantitative RT-PCR, Staining, Labeling, Fluorescence

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Guiqi Baizhu Decoction Alleviates Radiation Inflammation in Rats by Modulating the Composition of the Gut Microbiota

doi: 10.1155/2020/9017854

Figure Lengend Snippet: Effect of GQBZD on the inflammatory injury of intestinal in X-ray radiation rats. Histopathological observations of the colon stained with hematoxylin-eosin (a) were carried out under light microscopy with 40x magnification (scale bar 50 μ m). Colon pathology score was counted in microscope (b). Occludin ((c) and (d)), ZO-1 ((c) and (e)), P65 ((f) and (g)), and p-P65 ((f) and (h)) were tested by western blot and in comparison with the X-ray radiation group. Values are presented as mean ± SEM. Differences were assessed by ANOVA for multiple comparisons and denoted as follows: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ns P > 0.05.

Article Snippet: The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with Occludin antibody (GTX114949, GeneTex, Texas, USA), ZO-1 antibody (Ab96587, Cambridge, UK), P65 antibody (YT3108, ImmunoWay, Texas, USA), phosphorylated P65 antibody (B4568, ImmunoWay, Texas, USA), and GAPDH antibody (ab9485, Abcam, Cambridge, UK), respectively, for 2 h. After 1 h incubation with goat-anti-rabbit HRP-conjugated secondary antibody (ab97051, Abcam, Cambridge, UK), the protein bands were detected with luminal reagent (Millipore, Pittsburgh, USA) and their relative intensities were quantified using Image J.

Techniques: Staining, Light Microscopy, Microscopy, Western Blot, Comparison

Journal: International Journal of Molecular Medicine

Article Title: GFI1 promotes the proliferation and migration of esophageal squamous cell carcinoma cells through the inhibition of SOCS1 expression

doi: 10.3892/ijmm.2021.5017

Figure Lengend Snippet: GFI1 enhances NF-κB and STAT3 activity by inhibiting SOCS1 expression in esophageal squamous cell carcinoma cell lines. (A and B) siGFI1 was transfected into KYSE30 and KYSE150 cells. (A) mRNA and (B) protein expression levels of SOCS1 were examined using reverse transcription-quantitative PCR and western blotting, respectively. (C) NF-κB activity, (D) p-p65 and (E) p-STAT3 protein levels were analyzed in KYSE30/KYSE150 cells following transfection with siGFI1. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. si, small interfering RNA; GFI1, growth factor-independent 1; SOCS1, suppressor of cytokine signaling 1; p-, phosphorylated.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated (p)-p65 (1:1,000; cat. no. 310012) were obtained from Chengdu Zen Bioscience Co., Ltd. Rabbit monoclonal antibodies against STAT3 (1:1,000; cat. no. 4904S) and p-STAT3 (1:1,000; cat. no. 4113S) were purchased from Cell Signaling Technology, Inc.

Techniques: Activity Assay, Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: GFI1 promotes the proliferation and migration of esophageal squamous cell carcinoma cells through the inhibition of SOCS1 expression

doi: 10.3892/ijmm.2021.5017

Figure Lengend Snippet: SOCS1 inhibits the increase in esophageal squamous cell carcinoma cell proliferation and migration induced by GFI1. (A-H) siSOCS1 was transfected into the GFI1-knockdown KYSE30 and KYSE150 cells. (A) Colony formation assays were performed on the double transfected cells. (B) Cell migration was determined using Transwell assays. (C) NF-κB activity, (D) p-p65, p-STAT3 and SOCS1 protein levels were analyzed. (E-G) Ratio of p-p65 to p65 and STAT3 to p-STAT3, as well as (H) the semi-quantification of SOCS1 protein expression were determined using ImageJ. Magnification, ×200. * P<0.05, ** P<0.01 *** P<0.001 vs. control; # P<0.05, ## P<0.01 and ### P<0.001 vs. siGFI1. SOCS1, suppressor of cytokine signaling 1; si, small interfering RNA; GFI1, growth factor-independent 1; p-, phosphorylated.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated (p)-p65 (1:1,000; cat. no. 310012) were obtained from Chengdu Zen Bioscience Co., Ltd. Rabbit monoclonal antibodies against STAT3 (1:1,000; cat. no. 4904S) and p-STAT3 (1:1,000; cat. no. 4113S) were purchased from Cell Signaling Technology, Inc.

Techniques: Migration, Transfection, Knockdown, Activity Assay, Expressing, Control, Small Interfering RNA