Distribution and expression level of Cx43 in single and monolayer NRVMs transfected with different virus.

<p>(A) Single and confluent NRVMs with Cx43 overexpression or knockdown by adenovirus infection (m.o.i. = 15) for 48 h. The subcellular distribution of Cx43 in different groups as indicated was determined by immunostaining the cells with antibody specific for Cx43. Cell nucleus are indicated by Hoechest 33258 (1 µg/ml), and the arrows indicate the membrane-associated Cx43. (B, C) NRVMs were infected with adenovirus vector or adenovirus carrying Cx43 full sequences (wt-Cx43) or Cx43 siRNA (Cx43-KD, both m.o.i. = 15) for 48 h. The expression of Cx43 in NRVMs was determined in Triton X-100 soluble and insoluble fractions by Western blot (B) and normalized by the level of α-actin and then the level of Cx43 in control cells (C). The detection of GAPDH in non-junctional but not in junctional fraction represents a successful separation of non-junctional and junctional Cx43. ** denotes <i>P</i><0.01 <i>vs.</i> vector or scramble.</p

Role of Cx43 in LY uptake in NRVMs.

<p>(A, B) Typical confocal images of LY uptake in single and confluent NRVMs with Cx43 overexpression or silence by adenovirus infection (m.o.i. = 15). The LY uptake cells in each group as indicated were counted and expressed with percentage of the total recorded cells determined by 5–6 independent experiments for each bar. ** represents <i>P</i><0.01 <i>vs.</i> scramble (Scram) cells. (C) Corresponding alterations of global Ca²⁺ transients and LY uptake in response to manipulations of Cx43 in both single and confluent monolayer NRVMs. (D) HEK293 cells were transfected with vector or plasmids carrying Cx43 for 48 h and determined their levels of Cx43 expression by Western blot. (E, F) Statistical data of the LY uptake percentage and Ca²⁺ wave frequency from different groups of HEK293 cells as indicated were obtained from 5–6 independent experiments. ** denotes <i>P</i><0.01 <i>vs.</i> vector in all panels.</p

Impairment of dye uptake by gap junction inhibitors in NRVMs.

<p>(A) Typical confocal images of Lucifer yellow (LY) uptake in different groups of single and confluent NRVMs treated with DMSO or heptanol (1 or 1.5 mM) for 2 min, Gap 27 (300 µM) for 30 min and FFA (25 µM) for 5 min as indicated. (B, C) Statistical data were obtained from 6–8 independent determinations for each bar. * and ** represent <i>P</i><0.05 and <i>P</i><0.01, <i>vs.</i> DMSO, respectively.</p

Role of Cx43 in gap-mediated Ca²⁺ transients in NRVMs.

<p>(A) NRVMs were infected with adenovirus vector or adenovirus carrying Cx43 full sequences (wt-Cx43) or Cx43 siRNA (m.o.i. = 15) for 48 h. The expression of Cx43 in NRVMs was determined by Western blot, which was normalized by the level of GAPDH. ** denotes <i>P</i><0.01 vs. control. (B) Typical traces represent spontaneous Ca²⁺ transients in single and monolayer NRVMs with Cx43 overexpression or knockdown as indicated, and (C) Their statistical data of the transient frequency and amplitude were obtained from 5–6 independent experiments. (D) The statistical data of the dose-dependent effect of Cx43 deficiency on Ca²⁺ transients in single cells were obtained from 7–9 independent experiments. ** represents <i>P</i><0.01 <i>vs.</i> scramble (Scram) for each panel.</p

Pathways involved in the Cx43-associated mediation of Ca²⁺ activities in single and monolayer NRVMs.

<p>(A) The summarized data of the effects of nifedipine (Nif 1 µM, 10 min), ryanodine (Rya 100 µM, 10 min) and xestospongin C (XeC 10 µM, 20 min) on the LY uptake and intracellular Ca²⁺ frequency in NRVMs. (B) Typical traces represent the spontaneous Ca²⁺ transients in single and monolayer NRVMs prior to (Con) and after 2-APB (2 µM) for 10 min treatment. (C, D) The statistical data of the concentration-dependent effects of 2-APB on LY loading and Ca²⁺ transient frequency in single and confluent NRVMs as indicated were obtained from 8–12 determinations for each bar. **<i>P</i><0.01 <i>vs.</i> DMSO in each panel; ^##<i>P</i><0.01 <i>vs.</i> DMSO treated single cells.</p

Effect of gap inhibition on Ca²⁺ sparks in NRVMs and adult mouse cardiomyocytes.

<p>(A) Typical linescan images of Ca²⁺ sparks (upper panel) and their <i>F/F₀</i> changes over the time (lower panel) in single NRVM from different groups as indicated. (B, C, D) Statistical data of the spark frequency, amplitude and duration in NRVMs in different groups as indicated were obtained from 4–5 independent determinations, n = 20–36 cells for each panel. ** represents <i>P</i><0.01 <i>vs.</i> scramble. (E) Typical linescan images of Ca²⁺ sparks (upper panel) and their <i>F/F₀</i> changes over the time (lower panel) in adult mouse ventricular myocytes. The cells were respectively treated with vehicle (Con), 1.5 mM heptanol for 2 min, 300 µM Gap 27 for 30 min or 25 µM FFA for 5 min. (F) Statistical data from 3–5 independent determinations (n = 15–20 cells for each bar) show the effect of the drugs on the Ca²⁺ spark rate. ** represents <i>P</i><0.01 <i>vs.</i> control.</p

Additional file 1 of CXCL12/CXCR7/β-arrestin1 biased signal promotes epithelial-to-mesenchymal transition of colorectal cancer by repressing miRNAs through YAP1 nuclear translocation

Additional file 1: Fig. S1. A, B RT-qPCR and Western blot analysis of the expression of CXCR7 at mRNA and protein levels in CRC cells. Fig. S2. Transwell assay was performed in HCT116CXCR7 and SW620CXCR7 cells compared with controls. Migrating cells were quantified. **P < 0.01. ***P < 0.001. Fig. S3. The correlations of CXCR7 (ACKR3) with ZEB1 and SNAI1 were determined in CRC tissues by Gene Expression Profiling Interactive Analysis (GEPIA). Fig. S4. A, B RT-qPCR analysis of the levels of miR-124-3p and miR-188-5p in HCT116, HT29 and SW620 cells transfected with these miRNA mimics (124m, 188m) and inhibitors (124i, 188i). Fig. S5. A, B Western blot analysis of the expression levels of YAP1, DCLK1 and Vimentin in HCT116 and SW620 cells transfected Flag-YAP-5SA compared with vector control. Anti-Flag antibodies were used to indicate the overexpression of Flag-YAP-5SA plasmid in CRC cells. Fig. S6. IF analysis of YAP1 nuclear translocation in HCT116 cells treated with CXCL12 (100 ng/ml) for different time course as indicated. YAP1 was labeled with Alexa Fluor® 488 donkey anti-rabbit secondary antibody. Nuclei were visualized with DAPI. Scale bars, 50 µm. Fig. S7. Western blot analysis of the expression levels of β-arrestin1 in HCT116 nuclear extracts stimulated by CXCL12 (100 ng/ml) for different time course as indicated. GAPDH and Histone H3 were used as cytoplasmic and nuclear loading control, respectively. Fig. S8. Transcriptional factor YY1 was predicted to bind to promoters of miR-124-3p and miR-188-5p by TransmiR 2.0 database. Fig. S9. Western blot analysis of the expression levels of YY1, DCLK1 and Vimentin in HCT116 and SW620 cells transfected with vector control and HA-YY1 plasmid. HA-tag was used to indicate the overexpression of YY1 in CRC cells. Fig. S10. The correlations of YAP1 with ZEB1 and SNAI1 were determined in CRC tissues by Gene Expression Profiling Interactive Analysis (GEPIA)

Additional file 2 of CXCL12/CXCR7/β-arrestin1 biased signal promotes epithelial-to-mesenchymal transition of colorectal cancer by repressing miRNAs through YAP1 nuclear translocation

Additional file 2: Table S1. The significantly upregulated genes in HCT116LV-CXCR7 vs. HCT116Control. Table S2. The significantly upregulated and downregulated miRNAs in HCT116LV-CXCR7 vs. HCT116Control. Table S3. The sequences of siRNAs and miRNAs. Table S4. The sequences of primers for RT-qPCR

A Comparison Study of Single-Echo Susceptibility Weighted Imaging and Combined Multi-Echo Susceptibility Weighted Imaging in Visualizing Asymmetric Medullary Veins in Stroke Patients

<div><p>Background</p><p>Asymmetric medullary veins (AMV) are frequently observed in stroke patients and single-echo susceptibility weighted imaging (SWI_s) is the main technique in detecting AMV. Our study aimed to investigate which echo time (TE) on single-echo susceptibility is the optimal echo for visualizing AMV and to compare the ability in detecting AMV in stroke patients between SWI_s and multi-echo susceptibility weighted imaging (SWI_c).</p><p>Materials and Methods</p><p>Twenty patients with middle cerebral artery stroke were included. SWI was acquired by using a multi-echo gradient-echo sequence with six echoes ranging from 5 ms to 35.240 ms. Three different echoes of SWI_s including SWI_s1 (TE = 23.144 ms), SWI_s2 (TE = 29.192 ms) and SWI_s3 (TE = 35.240 ms) were reconstructed. SWI_c was averaged using the three echoes of SWI_s. Image quality and venous contrast of medullary veins were compared between SWI_s and SWI_c using peak signal-to-noise ratio (PSNR), mean opinion score (MOS), contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR). The presence of AMV was evaluated in each SWI_{s (1–3)} and SWI_c.</p><p>Results</p><p>SWI_s2 had the highest PSNR, MOS and CNR and SWI_s1 had the highest SNR among three different echoes of SWI_s. No significant difference was found in SNR between SWI_s1 and SWI_s2. PSNR, MOS and CNR in SWI_c were significantly increased by 27.9%, 28.2% and 17.2% compared with SWI_s2 and SNR in SWI_c was significantly increased by 32.4% compared with SWI_s1. 55% of patients with AMV were detected in SWI_s2, SWI_s3 and SWI_c, while 50% AMV were found in SWI_s1.</p><p>Conclusions</p><p>SWI_s using TE around 29ms was optimal in visualizing AMV. SWI_c could improve image quality and venous contrast, but was equal to SWI_s using a relative long TE in evaluating AMV. These results provide the technique basis for further research of AMV in stroke.</p></div

Description of contrast-to-noise ratio (CNR) measurement for deep medullary veins (DMV) in a combined multi-echo susceptibility weighted image.

<p>A, Each red region of interest (ROI) for periventricular DMV was drawn with one pixel width along the center of vein. Each blue ROI for neighboring white matter tissues was drawn surrounding the vein by the same pixel size of red ROI. Three different regions of DMV and adjacent white matter tissues (yellow rectangle in A) were shown (B to D, magnification of the three regions in A). CNR was calculated based on values obtained from the two ROIs.</p