Overexpression of GITR impacts TNFα-induced non-canonical NF-κB activation.

<p>A. MM1.S, OPM1 (GITR low) and RPMI8226 (GITR high) cells were exposed to TNFα (10ng/ml) for 60 minutes. NF-κB activity was evaluated by DNA binding ELISA assay. NF-κB p65 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experiments. *P<0.05, **P<0.01 and ***P<0.001 compared with indicated groups. B. MM1.S, OPM1 and RPMI8226 cells were exposed to TNFα (10ng/ml) for 60 minutes. Nuclear and whole cell lysates were subjected to western blot using anti-p65, -p52 and Actin antibodies. C. Cells were exposed to TNFα (10ng/ml) for 15, 30, or 60 minutes. Nuclear protein and cytoplasmic extraction were subjected to western blot using anti-p52, -RelB and -nucleolin antibodies. D. pCMV-GITR and GITR-MM1.S cells were harvested at 24 hours after treatment with and without TNF-α (10ng/ml) for 60 minutes. Immunocytochemical analysis was assessed using anti-phospho-NF-κB-p52 antibody, with DAPI used to stain nuclei.</p

Expression of GITR in MM patients.

<p>mRNA of GITR from 16 MM patients and 20 pooled normal bone marrow specimens were assessed by real time-PCR. 18S was considered as the internal control.</p

GITR expression correlates with NF-κB activation and sensitivity to NF-κB inhibitor.

<p>A. Sensitivity of MM cells to NF-κB inhibitor-BAY-11-7085 was assessed in five MM cell lines. Cell viability was determined by CellTiter-Glo assay. Veh indicating DMSO treated cells. Com indicates combination treatment of TNF with BAY-11-7085. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with Veh groups. B. MM cell lines were exposed to 10ng/ml TNF with/without BAY-11-7085. Cells were lysed after 12 hours incubation and subjected to Immunoblotting using anti-PARP1 and Actin antibodies.</p

Overexpression of GITR enhanced sensitivity to Bortezomib-induced apoptosis in MM1.S cells.

<p>A. Empty vector and GITR-transfected MM1.S cells were treated with Bortezomib for 48 hours. Cell viability was assessed by CellTiter-Glo assay. Data represent mean ± SD, **P<0.01 compared with indicated groups. B. Empty control and GITR expressing MM1.S cells were exposed to different doses of Bortezomib and incubated overnight. Cells were lysed and subjected to Immunoblotting using anti-PARP1 and Actin antibodies. C. Empty control and GITR expressing MM1.S cells were exposed to different doses of Bortezomib and incubated for 24 hours. The number of dead cells were assessed by PI single staining and quantified by Flowjo software. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with indicated groups. D. NF-κB activity was evaluated in control and GITR expressing MM1.S cells. Nuclear protein lysates were subjected to western blot using anti-p65 and nucleolin antibodies. E. Empty vector and GITR-transfected MM1.S cells were treated with 50nM Bortezomib for 48 hours in co-cultured with or without BMSC. After 48 hours incubation, the cell viability was assessed by CellTiter-Glo assay. Data represent mean ± SD, *P<0.05, **P<0.01 and ***P<0.001 compared with indicated groups.</p

Knockdown of GITR reduced the sensitivity to Bortezomib in RPMI cell line.

<p>A. Knockdown efficacy of GITR gene was assessed by real time-PCR. Scr (Scramble) indicates the non-targeting SiRNA control. B. Knockdown of GITR reduced sensitivity to Bortezomib in RPMI cell line. Cell viability was evaluated by CellTiter-Glo assay after 48 hours incubation. Data represent mean ± SD, * P<0.05, and **P<0.01. C. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated overnight. Cells were lysed and subjected to Immunoblotting using anti-cleaved caspase-3 and Actin antibodies. D. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated for 24 hours. The number of dead cells were assessed by FACS based PI single staining and quantified by Flowjo software. Data represent mean ± SD, * P<0.05, and **P<0.01 compared with indicated groups.</p

Overexpression of GITR enhanced Bortezomib induced tumor growth inhibition in MM1.S xenograft mice.

<p>A. Empty control and GITR expressing cells were isolated from MM1.S xenograft mice and subjected to mRNA extraction. The expression of GITR was examined by real time-PCR. GAPDH was considered as an internal control. B. CD138⁺ human plasma cells were isolated from femur of the four groups of investigated mice. The number of CD138⁺ cell was assessed by flow cytometry. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with indicated groups. C. NF-κB activity was evaluated by DNA binding ELISA assay. NF-κB p65 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experiments. ***P<0.001 compared with indicated groups.</p

The combination of chelerythrine and erlotinib significantly induced apoptosis in SK-MES-1 (Fig. 4A) and A549 (Fig. 4B) cells.

<p>Cells were harvested after treatment for 24 hours, washed twice with cold PBS, and re-suspended in a 500μl binding buffer for a final cell concentration of 1-5x10⁵cells/ml. They were incubated in the dark with 5μl of FITH Annexin V and 5 μlPI (20 μg/ml) for 30min. Subsequently, the suspension was analyzed by FCM. N = 3, Mean ± SD, **: P<0.01.</p

Effects of chelerythrine and erlotinib on SK-MES-1 xenograft mice model.

<p>A, B and C. NOD SCID mice were injected with 4×10⁶cells suspended in 0.1ml of PBS subcutaneously into the left chest. Treatments (erlotinib: 50mg/kg, o. p. daily; cheleryhine; 10mg/kg, o. p. daily; or a combination of both drugs; and DMSO was used as the control group) with drugs were initiated when the tumor volume reached about 150mm³. Tumor volume and mice weight were recorded every two days. At the end of the 14 day treatment, the mice were sacrificed, and the xenograft tumors were taken out and weighed. N = 6; Mean ± SEM; (E+C vs Control was considered very significant at Day14, **p = 0.0012; Che vs Control was also significant, *p<0.05; Two-way ANOVA). D. mice weights were monitored during the treatment. E. normal histologically appearance on the organs from a representative mouse treated with a combination of chelerythrine and erlotinib.</p

The combination treatment of chelerythrine and erlotinib significantly reduced cell migration and invasion in SK-MES-1(Fig. 3A & B) and A459 (Fig. 3C & D) cells.

<p>Viable cells were collected and counted after being treated with erlotinib, chelerythrine or a combination for 24h. The 4×10⁵ cells were plated into each insert and cultured for 20 hours. Cells were fixed and stained immediately. A and B: Migration and invasion of SK-MES-1 cells. D and E: Migration and invasion of A549 cells. N = 3, Mean ± SD, *: P<0.05, **: P<0.01.</p

Effects of erlotinib (Erl) or/and cherlerythrine (Che) on the viability of NSCLC cells.

<p>A to D: IC₅₀ of both compounds on HCC827, SK-MES-1 and A549 cells was assessed by alamar blue assay at 48 hours after drug treatment as described in the methods section. After IC₅₀ of each compound was identified, the combination effect on cell viability was assessed on erlotinib less sensitive SK-MES-1 and A549 cells at 24, 48 and 72 hours after treatment. E and F: The combination effect on SK-MES-1 cell growth. G and H: The combination effect on A549 cell growth. The fluorescence value was recorded at a range from 540nm to 590nm. The percentage of cell growth was calculated as following: cell growth (%) = (experiment well/control well) x 100%; n = 3. Mean ± SD. N = 3.</p