Figure 2
Figure 2. Knockdown of Fyn slows cell growth, reduces clonogenicity, inhibits growth of myeloid cells in vivo, and sensitizes cells to imatinib. (A) Cells expressing Fyn shRNA achieve knockdown of Fyn but not Lyn or c-abl. K562 cells were stably transfected with Fyn shRNA as described in “Design of shRNA to Fyn.” Levels of Fyn (top panel), Lyn, and c-abl (bottom panel) were assessed by Western blotting in cells transfected with Fyn shRNA plasmid mix (left panels) or lentivirally expressed shRNAs (U613 + U67). Numeric values indicate the ratio of Fyn/actin normalized to the scrambled/noncoding shRNA-expressing K562 cells. In the left bottom panel, Fyn mRNA was detected by RT-PCR in K562 cells transfected with scrambled shRNA or shRNA plasmids directed toward Fyn. Numeric values represent the ratio of band intensity corresponding to Fyn mRNA to actin band intensity and are not normalized. Right panel shows a kinase assay for BCR-ABL1 in K562 cells infected with nonspecific shRNA (LV) or Fyn-targeted shRNAs (U613 + U67). Phosphorylation of the BCR-ABL1 substrate, p-Crk, is lost in Fyn shRNA–expressing cells. (B) Fyn knockdown slows cell growth significantly. K562 cells transfected with scrambled shRNA or Fyn shRNA (left graph) were plated at a density of 200 000 cells/mL. After 24 hours and 48 hours, the numbers of cells were counted using a ViCell Coulter Counter (Beckman Coulter). At 48 hours, there was a statistically significant difference (P < .01) in cell number. Overexpression of a mutant Fyn not degraded by Fyn shRNA restores cell numbers (right graph). K562 cells were plated at a density of 750 000 cells/mL, and cell numbers were counted at 24 and 48 hours. At 48 hours, there was a statistically significant difference in cell number (P < .04). (C) Fyn knockdown significantly diminishes clonogenic growth. K562 cells infected with nothing (K562), lentivirus with nonspecific shRNA (LV), lentivirus with Fyn shRNAs (U613 + U67), or mutant Fyn not degraded by shRNA were plated in Methocult media, and the numbers of colonies were counted after 5 days. There was a significant decrease in colony number in Fyn shRNA–expressing cells (P < .05), whereas the rescue construct restored clonogenic potential (P < .05). (D) Fyn knockdown sensitizes cells to imatinib-induced apoptosis. K562 cells infected with nothing (K562), lentivirus with nonspecific shRNA (LV), lentivirus with Fyn shRNAs (U613 + U67), or mutant Fyn not degraded by shRNA were treated with 5 μM imatinib for 48 hours. Subdiploid percentage of cells, indicative of DNA fragmentation, was assessed by PI staining and subsequent analysis by flow cytometry. Bar graph depicts 3 experiments. (E) Myeloid cell growth in vivo is slowed in SCID mice injected with Fyn shRNA–expressing cells. SCID mice were injected with 20 million K562 cells expressing scrambled or Fyn-directed shRNA. Injected cells were more than 90% viable as determined by trypan blue staining. After 4 weeks, peripheral blood from 10 mice (5 reconstituted with scrambled shRNA and 5 reconstituted with Fyn shRNA) was collected and CBCs were conducted. Percentages of segmented cells are shown. Error bars represent SD. (F) Survival of SCID mice reconstituted with Fyn shRNA–expressing cells versus scrambled shRNA–expressing cells. Kaplan-Meier analysis of mice described in panel 2E (P = .079).

Knockdown of Fyn slows cell growth, reduces clonogenicity, inhibits growth of myeloid cells in vivo, and sensitizes cells to imatinib. (A) Cells expressing Fyn shRNA achieve knockdown of Fyn but not Lyn or c-abl. K562 cells were stably transfected with Fyn shRNA as described in “Design of shRNA to Fyn.” Levels of Fyn (top panel), Lyn, and c-abl (bottom panel) were assessed by Western blotting in cells transfected with Fyn shRNA plasmid mix (left panels) or lentivirally expressed shRNAs (U613 + U67). Numeric values indicate the ratio of Fyn/actin normalized to the scrambled/noncoding shRNA-expressing K562 cells. In the left bottom panel, Fyn mRNA was detected by RT-PCR in K562 cells transfected with scrambled shRNA or shRNA plasmids directed toward Fyn. Numeric values represent the ratio of band intensity corresponding to Fyn mRNA to actin band intensity and are not normalized. Right panel shows a kinase assay for BCR-ABL1 in K562 cells infected with nonspecific shRNA (LV) or Fyn-targeted shRNAs (U613 + U67). Phosphorylation of the BCR-ABL1 substrate, p-Crk, is lost in Fyn shRNA–expressing cells. (B) Fyn knockdown slows cell growth significantly. K562 cells transfected with scrambled shRNA or Fyn shRNA (left graph) were plated at a density of 200 000 cells/mL. After 24 hours and 48 hours, the numbers of cells were counted using a ViCell Coulter Counter (Beckman Coulter). At 48 hours, there was a statistically significant difference (P < .01) in cell number. Overexpression of a mutant Fyn not degraded by Fyn shRNA restores cell numbers (right graph). K562 cells were plated at a density of 750 000 cells/mL, and cell numbers were counted at 24 and 48 hours. At 48 hours, there was a statistically significant difference in cell number (P < .04). (C) Fyn knockdown significantly diminishes clonogenic growth. K562 cells infected with nothing (K562), lentivirus with nonspecific shRNA (LV), lentivirus with Fyn shRNAs (U613 + U67), or mutant Fyn not degraded by shRNA were plated in Methocult media, and the numbers of colonies were counted after 5 days. There was a significant decrease in colony number in Fyn shRNA–expressing cells (P < .05), whereas the rescue construct restored clonogenic potential (P < .05). (D) Fyn knockdown sensitizes cells to imatinib-induced apoptosis. K562 cells infected with nothing (K562), lentivirus with nonspecific shRNA (LV), lentivirus with Fyn shRNAs (U613 + U67), or mutant Fyn not degraded by shRNA were treated with 5 μM imatinib for 48 hours. Subdiploid percentage of cells, indicative of DNA fragmentation, was assessed by PI staining and subsequent analysis by flow cytometry. Bar graph depicts 3 experiments. (E) Myeloid cell growth in vivo is slowed in SCID mice injected with Fyn shRNA–expressing cells. SCID mice were injected with 20 million K562 cells expressing scrambled or Fyn-directed shRNA. Injected cells were more than 90% viable as determined by trypan blue staining. After 4 weeks, peripheral blood from 10 mice (5 reconstituted with scrambled shRNA and 5 reconstituted with Fyn shRNA) was collected and CBCs were conducted. Percentages of segmented cells are shown. Error bars represent SD. (F) Survival of SCID mice reconstituted with Fyn shRNA–expressing cells versus scrambled shRNA–expressing cells. Kaplan-Meier analysis of mice described in panel 2E (P = .079).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal