Figure 4
Figure 4. Gfi1, a transcriptional repressor, is induced in BMSCs and MC-4 cells by MM cells and TNF-α. (A) Primary BMSCs samples, as described in Figure 1, were analyzed for Gfi1 mRNA levels relative to GAPDH by quantitative PCR. (B) RNA from MC-4 cells cocultured with 5TGM1 cells (in transwell inserts) for the indicated times (h) before removal of the 5TGM1 cells and RNA isolation from the MC-4 cells. Expression of Gfi1 was detected by quantitative PCR relative to GAPDH and the “MC-4 alone” sample. (C) MC-4 cells were treated with 1 ng/mL TNF-α or IL-7 for 6 hours, and Gfi1 mRNA expression was determined by quantitative PCR relative to GAPDH. (D) Western blot analysis of Gfi1 protein expression and localization using whole cell, cytoplasmic, and nuclear extracts from 5TGM1 cells, MC-4 cells, and MC-4 cells treated with 5TGM1 cells in a transwell, TNF-α (1 ng/mL), or IL-7 (2 ng/mL). For loading controls, the blots were probed with anti–α-tubulin, TFIIB, or β-actin as indicated. Note that anti–TFIIB did not detect anything in the cytoplasmic extracts and anti–α-tubulin did not detect anything in the nuclear extracts, indicating that there was no significant cross-contamination of the subcellular compartments during their preparation. (E) The BM aspirates from MM patients and normals were cultured overnight; then the adherent cells were CD138-depleted using negative immunoselection (MACS, Miltenyi) and the BMSCs expanded for 3 weeks before use. Stromal cells obtained from 4 normals (●) and 11 MM patients (▴) were analyzed for Gfi1 expression by quantitative PCR relative to GAPDH and the fold change plotted with the SEM. Gfi1 RNA expression was significantly increased with a mean of 8.4-fold in the BMSCs from MM patients compared with the stromal cells from normals (P < .0006) using a 2-tailed unpaired t test with Welch correction. The Gfi1/GAPDH range for the normals was 0.24 to 1.23 and for the MM patients was 0.84 to 8.04 with the fold change MM relative to normal calculated among samples run at the same time. The 11 patients all had bone disease and stage 3 MM; 6 were newly diagnosed and 5 were relapsed, and there was no difference in the Gfi1 levels between these groups. (F) Western blot analysis for human Gfi1 protein expression in the BMSCs from 3 MM patients and 1 normal, along with Jurkat extract as a positive control (20 μg each). The ratio of Gfi1 to β-actin was calculated for each sample and noted below the blot. The ratio of Gfi1 to β-actin for these samples plus data from another 5 MM patients and 4 normals are plotted with the mean and SEM indicated. Gfi1 protein expression was significantly increased with a mean of 18-fold in the BMSCs from MM patients compared with the stromal cells from normals (P < .0194) using a 2-tailed unpaired t test.

Gfi1, a transcriptional repressor, is induced in BMSCs and MC-4 cells by MM cells and TNF-α. (A) Primary BMSCs samples, as described in Figure 1, were analyzed for Gfi1 mRNA levels relative to GAPDH by quantitative PCR. (B) RNA from MC-4 cells cocultured with 5TGM1 cells (in transwell inserts) for the indicated times (h) before removal of the 5TGM1 cells and RNA isolation from the MC-4 cells. Expression of Gfi1 was detected by quantitative PCR relative to GAPDH and the “MC-4 alone” sample. (C) MC-4 cells were treated with 1 ng/mL TNF-α or IL-7 for 6 hours, and Gfi1 mRNA expression was determined by quantitative PCR relative to GAPDH. (D) Western blot analysis of Gfi1 protein expression and localization using whole cell, cytoplasmic, and nuclear extracts from 5TGM1 cells, MC-4 cells, and MC-4 cells treated with 5TGM1 cells in a transwell, TNF-α (1 ng/mL), or IL-7 (2 ng/mL). For loading controls, the blots were probed with anti–α-tubulin, TFIIB, or β-actin as indicated. Note that anti–TFIIB did not detect anything in the cytoplasmic extracts and anti–α-tubulin did not detect anything in the nuclear extracts, indicating that there was no significant cross-contamination of the subcellular compartments during their preparation. (E) The BM aspirates from MM patients and normals were cultured overnight; then the adherent cells were CD138-depleted using negative immunoselection (MACS, Miltenyi) and the BMSCs expanded for 3 weeks before use. Stromal cells obtained from 4 normals (●) and 11 MM patients (▴) were analyzed for Gfi1 expression by quantitative PCR relative to GAPDH and the fold change plotted with the SEM. Gfi1 RNA expression was significantly increased with a mean of 8.4-fold in the BMSCs from MM patients compared with the stromal cells from normals (P < .0006) using a 2-tailed unpaired t test with Welch correction. The Gfi1/GAPDH range for the normals was 0.24 to 1.23 and for the MM patients was 0.84 to 8.04 with the fold change MM relative to normal calculated among samples run at the same time. The 11 patients all had bone disease and stage 3 MM; 6 were newly diagnosed and 5 were relapsed, and there was no difference in the Gfi1 levels between these groups. (F) Western blot analysis for human Gfi1 protein expression in the BMSCs from 3 MM patients and 1 normal, along with Jurkat extract as a positive control (20 μg each). The ratio of Gfi1 to β-actin was calculated for each sample and noted below the blot. The ratio of Gfi1 to β-actin for these samples plus data from another 5 MM patients and 4 normals are plotted with the mean and SEM indicated. Gfi1 protein expression was significantly increased with a mean of 18-fold in the BMSCs from MM patients compared with the stromal cells from normals (P < .0194) using a 2-tailed unpaired t test.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal