Expression of CGRP among all groups was detected by immunocytochemistry.

<p><b>A), B)</b> Expression of the CGRP gene in the transduced group showed higher secretion of CGRP protein than in the control group after 3days and 7days. Scale bar equals 100 µm. <i>*p</i><0.05, transduced group vs. control group.</p

Radiography observation in bone defect model.

<p><b>A)</b> X-rays photograph of rat radical defects in different groups at 2 weeks, 4 weeks, and 12 weeks showing complete bridging of the defect in group 3, however, a radiolucent line was still present at the defect site in group 1 at 12 weeks. <b>B)</b> Radiographical scoring also showed that new bone formation in group 3 was more significant difference than the other groups at 12 weeks (p<0.05). (Group 1 referred to <i>β</i>-TCP group; Group 2 referred to ADSCs/<i>β</i>-TC group; Group 3 referred to Ad-CGRP-ADSCs/<i>β</i>-TCP group).</p

Levels of ALP (U/L, ± s) in transduced and control cells.

*<p><i>p<0.05,</i> transduced group vs. control group.</p

Mineralized nodule formation after osteoblast induction.

<p>Mineralized nodule formation was detected on days 7 and 14. The transduced group indicated more mineralized nodules which were display as circular, golden brown nodules formed from the combination of f tetracycline and calcium. Scale bar equals 100 µm.</p

Histological observation in bone defect model.

<p>HE staining of the transverse bone defect sections in different groups at 4 weeks and 12 weeks post-surgery. Abbreviations used: scaffold (S); new bone from implanted scaffold (NB-S); bone marrow (BM); cortical bone (CB), trabecular bone (TB). Scale bar equals 400µm. (Group 1 referred to <i>β</i>-TCP group; Group 2 referred to ADSCs/<i>β</i>-TC group; Group 3 referred to Ad-CGRP-ADSCs/<i>β</i>-TCP group).</p

Expression of CGRP among all groups was detected by RT-PCR and Western blot.

<p><b>A), B)</b> demonstrated higher expression of CGRP in the transduced group than the control group at 3 d or 7 d after transduction by RT-PCR and Western blot detections. <i>*p</i><0.05, transduced group vs. control group.</p

Expression of ALP after osteoblast induction.

<p>Detection of ALP staining in the transduced group displayed more red ALP-positive granules in the cytoplasm than the control group on days 7 and 14. Scale bar equals 100 µm.</p

Transduction efficiency and growth curves of ADSCs.

<p><b>A)</b> CGRP-transduced ADSCs exhibited bright green EGFP fluorescence under fluorescent microscope and indicated a transduction efficiency of approximately 81%. Scale bar equals 100 µm. <b>B)</b> The growth curves of different groups showed that the transduced group had a significantly shorter doubling time than the untransduced group. <i>*p</i><0.05, transduced group vs. control group.</p

Primers used for real-time PCR analysis.

<p>Primers used for real-time PCR analysis.</p

Additional file 1: Figure S1. of Inhibition of EGR1 inhibits glioma proliferation by targeting CCND1 promoter

Identification and characterization of U251 stem-like cells(USLC). Figure S2. Schematic diagram of the EGR1 binding site in CCND1 promotor. Figure S3. The mRNA expression of EGR1 and CCND1 at different points in time after adding EGF in U251 cells. Figure S4. The another two databases apart from JASPAR to corroborate the EGR1 binding site. Figure S5. Negative controls for the immunohistochemistry. Figure S6 GAPDH promoter is regulated by EGR1. Figure S7. The positive expression control for EGR1, to validate the antibody. (DOC 2277 kb