Journal:

Article Title: Effects of CXCR4 Gene Transfer on Cardiac Function After Ischemia-Reperfusion Injury

doi: 10.2353/ajpath.2010.090451

Figure Lengend Snippet: CXCL12 is upregulated in ischemic myocardium of CXCR4-overexpressed rat. A: CXCL12 protein expression was assessed in hearts injected with either CXCR4 or β-gal and/or control (saline-injected; with IR) by immunofluorescence staining. Frozen sections were fixed and stained with anti-CXCL12 (green) and anti–α actinin (red). Primary Abs visualized with FITC or Texas Red conjugate. Nuclei were stained with DAPI. Images are taken with confocal microscopy. B: Protein lysates were prepared from rats’ hearts one week after CXCR4 gene transfer followed by 30 minutes LAD ligation and 24 hours reperfusion. The ischemic and remote tissues were lysed and analyzed by Western blot. Representative gel of three independent experiments is shown. C: CXCL12 mRNA levels were determined by quantitative real-time PCR (QRT-PCR) using a QuantiTect SYBR Green RT-PCR Kit and using specific primers for CXCL12 and 18S. Primers were designed to generate short amplification products. Densitometric analysis of data from three different experiments is shown. *P < 0.05.

Article Snippet: The following antibodies were used: (1) rabbit anti-mouse/rat CXCL12 (eBiosciences; San Diego, CA); (2) goat anti-rat CXCR4 antibody (Torrey Pines Biolabs; East Orange, NJ); (3) goat anti-mouse/rat TNF- α, (R&D), (4) mouse anti-rat leukocyte antibody (BD Pharmingen; Franklin Lakes, NJ); and (5) mouse anti- rat macrophage marker (VP-M640, Vector Laboratories; Burlingame, CA) Sections from the hearts were fixed and blocked with the normal serum and incubated with primary antibody for 2 hours at room temperature.

Techniques: Expressing, Injection, Immunofluorescence, Staining, Confocal Microscopy, Ligation, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Molecular Therapy Oncolytics

Article Title: COMMD7 Regulates NF-κB Signaling Pathway in Hepatocellular Carcinoma Stem-like Cells

doi: 10.1016/j.omto.2018.12.006

Figure Lengend Snippet: Overexpression of PIAS4 Influenced the Effect of COMMD7 Knockdown in Nanog + HCSCs (A and B) The expression of PIAS4 was determined using real-time qPCR (A) and western blotting (B) analysis of the protein level of CXCL12, CXCL2, and PIAS4 in Nanog + HCSCs after COMMD7 knockdown. Then the COMMD7-silenced Nanog + HCSCs were transfected with empty vector or pcDNA3.1-PIAS4. (C) The mRNA expression of PIAS4 after PIAS4 overexpression. (D) Western blotting analysis of the protein expression of NEMO, p65, HNF4α, and PIAS4. The proliferative ability, apoptotic rate, migration, and invasion were evaluated using (E) CCK-8 assay, (F) flow cytometry assay, and (G) wound healing and transwell invasion assay in Nanog + HCSCs, respectively. *p < 0.05; ***p < 0.001 versus vector or sh-COMMD7.

Article Snippet: Next, the membranes were blocked in 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature followed by incubation with primary antibodies against Nanog (1:1,000, ab153419; Abcam), COMMD7 (1:2,000, #9742; Cell Signaling), COMMD1 (1:1,000, #24640; SAB), NF-κB p65 (1:1,500, 19553-1-AP; Proteintech), HNF4α (1:1,000, #2947; Cell Signaling), NEMO (1:3,000, #23870; SAB), CXCL12 (1:1,000, #36120; SAB), CXCL2 (1:4,000, #24681; SAB), PIAS4 (1:2,500, #8709; Cell Signaling), histone H3.1 (1:2,000, #9265; Cell Signaling), β-actin (1:1,000, #5762; Cell Signaling), and GAPDH (1:10,000, 10494-1-AP; Proteintech) at 4°C overnight.

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Migration, CCK-8 Assay, Flow Cytometry, Transwell Invasion Assay

Journal: Journal of Biomedical Research

Article Title: DEC1 promotes breast cancer bone metastasis through transcriptional activation of CXCR4

doi: 10.7555/JBR.39.20250031

Figure Lengend Snippet: DEC1 deficiency prevented breast bone metastasis induced by intracardiac injections of 4T1 cells via decreasing CXCR4/CXCL12 in mice. Forty 4-month-old mice, comprising twenty Dec1 +/+ and twenty Dec1 −/− , were divided into four groups: Dec1 +/+ -PBS, Dec1 +/+ -4T1, Dec1 −/− -PBS, and Dec1 −/− -4T1. Mice in the Dec1 +/+ -4T1 and Dec1 −/− -4T1 groups received an intracardiac injection of 4T1 mouse breast cancer cells to induce breast bone metastasis, while mice in the Dec1 +/+ -PBS and Dec1 −/− -PBS groups received an equal volume of PBS via the same method for two months. n = 10 for each group. A: Osteolytic bone injury in the four groups of mice by X-ray. B: The serum CXCL12 amount in the four groups of mice ( n = 3–5). C–F: The CA153, CK8, and Ki67 expression in the four groups of mice by immunohistochemical staining ( n = 3). G and H: The TRAP-positive cells in the four groups of mice ( n = 3). I–N: The protein levels of CXCR4, TRAP, N-cadherin, E-cadherin, and vimentin in the four groups of mice by Western blotting ( n = 3). O: The CXCR4 expression in the four groups of mice. Data are presented as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns P > 0.05, comparisons are shown in the figure. Data were analyzed using two-way ANOVA, and differences between groups were analyzed using Student's t -test.

Article Snippet: CXCL12 enzyme-linked immunosorbent assay (ELISA) Kit (Cat. #KE10046) was from Proteintech (Wuhan, China).

Techniques: Injection, Expressing, Immunohistochemical staining, Staining, Western Blot, Standard Deviation

Journal: Journal of Biomedical Research

Article Title: DEC1 promotes breast cancer bone metastasis through transcriptional activation of CXCR4

doi: 10.7555/JBR.39.20250031

Figure Lengend Snippet: DEC1 promoted CXCL12 production from mesenchymal stromal cells in mice and MC3T3-E1 cells. A: The CXCL12 expression in the femur of wildtype (WT) and Dec1 -knockout (KO) mice by immunohistochemical staining ( n = 3 in each group). B: The relative Cxcl12 mRNA levels in bone tissues from the femur of WT and Dec1 -KO mice ( n = 5 in each group). C: The relative Cxcl12 mRNA levels in mesenchymal cells from the femur of WT and Dec1 -KO mice ( n = 3 in each group). D–F: MC3T3-E1 cells were seeded into 6-well plates and cultured overnight. Cells were divided into three groups: Vector (infected with LV-shCon and transfected with Flag-CMV2), DEC1 -OE (transfected with Flag- DEC1 ), and DEC1 -KD (infected with LV-sh DEC1 ). After 24 h of transfection or infection, the culture medium from each group was collected, centrifuged, and filtered to obtain conditioned medium (CM). Effect of CM from MC3T3-E1 cells with DEC1 -OE or KD on breast cancer cell migration by Transwell migration assays (D). The relative Cxcl12 mRNA levels in MC3T3-E1 cells (E). The CXCL12 amount in CMs from DEC1 -OE and KO in MC3T3-E1 cells by ELISA assays (F). G: Mechanism of DEC1 promoting breast cancer bone metastasis via transcriptional activation of CXCR4. Data are presented as mean ± standard deviation (all experiments were repeated at least three times). * P < 0.05, ** P < 0.01, *** P < 0.001, comparisons are shown in the figure. Data were analyzed using one-way ANOVA, and differences between groups were analyzed using Student's t -test. Abbreviation: EMT, epithelial-to-mesenchymal transition; KD, knockdown; OE, overexpression.

Article Snippet: CXCL12 enzyme-linked immunosorbent assay (ELISA) Kit (Cat. #KE10046) was from Proteintech (Wuhan, China).

Techniques: Expressing, Knock-Out, Immunohistochemical staining, Staining, Cell Culture, Plasmid Preparation, Infection, Transfection, Migration, Enzyme-linked Immunosorbent Assay, Activation Assay, Standard Deviation, Knockdown, Over Expression