Figure 3.
Figure 3. Loss of HS inhibits MM cell growth by attenuating Wnt signaling. (A) Flow cytometry analysis of the effect of doxycycline (Dox)–induced EXT1 KD on MM cell expansion at 5 days of culture, relative to day 0. Short hairpin Scramble (shScramble) was used as control. The mean ± standard deviation (SD) of 3 independent experiments in triplicate is shown. *P ≤ .05, **P ≤ .01 using unpaired Student t test. (B) Flow cytometry analysis of primary MM cell-surface HS expression after heparitinase treatment. (C) Flow cytometry analysis of primary MM cell expansion after heparitinase or control buffer treatment. A representative plot for 2 independent experiments is shown. **P ≤ .01 using unpaired Student t test. (D) Flow cytometry analysis of the effect of Dox-induced EXT1 KD on HMCL apoptosis. The mean ± SD of 3 independent experiments in triplicate is shown, not significant (ns) using unpaired Student t test. (E) Left: representative plot of cell-cycle analysis after BrdU incorporation upon Dox-induced EXT1 KD in HMCLs. Right: quantification of cell-cycle distribution after BrdU incorporation in Dox-induced EXT1 KD in HMCLs, The mean ± SD of 3 independent experiments in triplicate is shown. *P ≤ .05, **P ≤ .01, ***P ≤ .001 using unpaired Student t test. (F) Flow cytometry analysis of the effect of Dox-induced EXT1 KD on the expansion of HMCLs in the presence of BM stromal cells (BMSCs) in 4 days. The mean ± SD of 3 independent experiments each performed in triplicate is shown. *P ≤ .05, **P ≤ .01 using 1-way analysis of variance (ANOVA) with Bonferroni correction. (G) Flow cytometry analysis of the effect of Dox-induced EXT1 KD on the expansion of HMCLs transduced with S33Y β-catenin or empty control vector in 5 days. The mean ± SD of 3 independent experiments in triplicate is shown. ns P > .05, *P ≤ .05, ***P ≤ .001 using 1-way ANOVA with Bonferroni correction. (H) Flow cytometry analysis of the effect of Dox-induced EXT1 KD on the expansion of HMCLs transduced with c-Myc or empty control vector in 5 days. The mean ± SD of 3 independent experiments in triplicate is shown. ns P > .05, **P ≤ .01, ***P ≤ .001 using 1-way ANOVA with Bonferroni correction.

Loss of HS inhibits MM cell growth by attenuating Wnt signaling. (A) Flow cytometry analysis of the effect of doxycycline (Dox)–induced EXT1 KD on MM cell expansion at 5 days of culture, relative to day 0. Short hairpin Scramble (shScramble) was used as control. The mean ± standard deviation (SD) of 3 independent experiments in triplicate is shown. *P ≤ .05, **P ≤ .01 using unpaired Student t test. (B) Flow cytometry analysis of primary MM cell-surface HS expression after heparitinase treatment. (C) Flow cytometry analysis of primary MM cell expansion after heparitinase or control buffer treatment. A representative plot for 2 independent experiments is shown. **P ≤ .01 using unpaired Student t test. (D) Flow cytometry analysis of the effect of Dox-induced EXT1 KD on HMCL apoptosis. The mean ± SD of 3 independent experiments in triplicate is shown, not significant (ns) using unpaired Student t test. (E) Left: representative plot of cell-cycle analysis after BrdU incorporation upon Dox-induced EXT1 KD in HMCLs. Right: quantification of cell-cycle distribution after BrdU incorporation in Dox-induced EXT1 KD in HMCLs, The mean ± SD of 3 independent experiments in triplicate is shown. *P ≤ .05, **P ≤ .01, ***P ≤ .001 using unpaired Student t test. (F) Flow cytometry analysis of the effect of Dox-induced EXT1 KD on the expansion of HMCLs in the presence of BM stromal cells (BMSCs) in 4 days. The mean ± SD of 3 independent experiments each performed in triplicate is shown. *P ≤ .05, **P ≤ .01 using 1-way analysis of variance (ANOVA) with Bonferroni correction. (G) Flow cytometry analysis of the effect of Dox-induced EXT1 KD on the expansion of HMCLs transduced with S33Y β-catenin or empty control vector in 5 days. The mean ± SD of 3 independent experiments in triplicate is shown. ns P > .05, *P ≤ .05, ***P ≤ .001 using 1-way ANOVA with Bonferroni correction. (H) Flow cytometry analysis of the effect of Dox-induced EXT1 KD on the expansion of HMCLs transduced with c-Myc or empty control vector in 5 days. The mean ± SD of 3 independent experiments in triplicate is shown. ns P > .05, **P ≤ .01, ***P ≤ .001 using 1-way ANOVA with Bonferroni correction.

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