Figure 3.
Figure 3. ATRA restores PU.1 expression in APL cells. (A) Northern blot analysis of HT93A cells treated with 1 μM ATRA and harvested at the time points indicated. The blot was probed for PU.1 (top), G-CSF receptor (middle), and GAPDH expression (bottom). (B) HT93A cells were treated with 1 μM ATRA. PU.1 mRNA expression was determined by quantitative real-time PCR. Results are given as fold induction of the PU.1/GAPDH ratio compared with the value of HT93A cells before treatment (0 hours). Mean values and SD (error bars) are depicted. (C) Western analysis using a RARA antiserum (top), a PU.1 antibody (second), an M-CSF receptor antibody (third), and a β-tubulin antiserum (bottom). HT93A cells were harvested before and after ATRA treatment for 24 hours. (D) EMSA of DNA-binding activity to the PU.1 site in the M-CSF receptor promoter. HT93A cells were stimulated with 1 μM ATRA and harvested at the time points indicated. ivt indicates in vitro-translated PU.1 protein; P, free probe alone; SS, supershift with specific antibody; PU.1, shift of PU.1 protein binding to this particular site; and X, nonspecific binding. The panel on the right represents binding of the same extracts to an NFY site as a control for loading and integrity of the extracts. N indicates treatment of the extracts with normal rabbit serum; and C, competition with cold oligo. (E) ATRA treatment overcomes the suppressive effect of PML-RARA on PU.1 expression. Northern blot showing PU.1 mRNA expression of U937-PML/RARA (U937-PR9) cells (top). Lane 1 depicts U937-PML-RARA cells in the absence of zinc or ATRA. Lane 2 shows U937-PR9 cells after 2 days of treatment with zinc (100 μM); lane 3 demonstrates U937-PR9 cells after 2 days of treatment with zinc (100 μM) and also treated with both ATRA (1 μM) and zinc for the last 24 hours. The same blot was probed for GAPDH (bottom). (F) Western blot analysis of whole-cell lysates of U937-PR9 cells. Lane 1 represents unstimulated cells. Cells were induced with zinc for 1 and 2 days (lanes 2 and 3). In lane 4 cells were treated for 48 hours with zinc, and then for 18 hours with both zinc and ATRA. Top: Staining with a RARA antibody detecting the fusion protein PML-RARA and the endogenous RARA. Middle: Probing the same blot with an antibody-detecting PU.1 protein. Bottom: The blot was restained for β-tubulin. (G) ATRA sensitivity is a prerequisite for PU.1 restoration in APL cells. Whole-cell lysates were analyzed by Western blot using a PU.1 antibody. Unstimulated U937-PR9 cells (lane 1) served as a positive control and Jurkat cells as a negative control (lane 3). Lane 2 shows U937-PR9 cells after 24 hours of PML-RARA induction. NB4 cells (lanes 4 to 6) and the ATRA-resistant APL cell line NB4-R1 (lanes 7 to 9) were induced for 12 or 24 hours with 1 μM ATRA. The blot was restained for β-tubulin (bottom).

ATRA restores PU.1 expression in APL cells. (A) Northern blot analysis of HT93A cells treated with 1 μM ATRA and harvested at the time points indicated. The blot was probed for PU.1 (top), G-CSF receptor (middle), and GAPDH expression (bottom). (B) HT93A cells were treated with 1 μM ATRA. PU.1 mRNA expression was determined by quantitative real-time PCR. Results are given as fold induction of the PU.1/GAPDH ratio compared with the value of HT93A cells before treatment (0 hours). Mean values and SD (error bars) are depicted. (C) Western analysis using a RARA antiserum (top), a PU.1 antibody (second), an M-CSF receptor antibody (third), and a β-tubulin antiserum (bottom). HT93A cells were harvested before and after ATRA treatment for 24 hours. (D) EMSA of DNA-binding activity to the PU.1 site in the M-CSF receptor promoter. HT93A cells were stimulated with 1 μM ATRA and harvested at the time points indicated. ivt indicates in vitro-translated PU.1 protein; P, free probe alone; SS, supershift with specific antibody; PU.1, shift of PU.1 protein binding to this particular site; and X, nonspecific binding. The panel on the right represents binding of the same extracts to an NFY site as a control for loading and integrity of the extracts. N indicates treatment of the extracts with normal rabbit serum; and C, competition with cold oligo. (E) ATRA treatment overcomes the suppressive effect of PML-RARA on PU.1 expression. Northern blot showing PU.1 mRNA expression of U937-PML/RARA (U937-PR9) cells (top). Lane 1 depicts U937-PML-RARA cells in the absence of zinc or ATRA. Lane 2 shows U937-PR9 cells after 2 days of treatment with zinc (100 μM); lane 3 demonstrates U937-PR9 cells after 2 days of treatment with zinc (100 μM) and also treated with both ATRA (1 μM) and zinc for the last 24 hours. The same blot was probed for GAPDH (bottom). (F) Western blot analysis of whole-cell lysates of U937-PR9 cells. Lane 1 represents unstimulated cells. Cells were induced with zinc for 1 and 2 days (lanes 2 and 3). In lane 4 cells were treated for 48 hours with zinc, and then for 18 hours with both zinc and ATRA. Top: Staining with a RARA antibody detecting the fusion protein PML-RARA and the endogenous RARA. Middle: Probing the same blot with an antibody-detecting PU.1 protein. Bottom: The blot was restained for β-tubulin. (G) ATRA sensitivity is a prerequisite for PU.1 restoration in APL cells. Whole-cell lysates were analyzed by Western blot using a PU.1 antibody. Unstimulated U937-PR9 cells (lane 1) served as a positive control and Jurkat cells as a negative control (lane 3). Lane 2 shows U937-PR9 cells after 24 hours of PML-RARA induction. NB4 cells (lanes 4 to 6) and the ATRA-resistant APL cell line NB4-R1 (lanes 7 to 9) were induced for 12 or 24 hours with 1 μM ATRA. The blot was restained for β-tubulin (bottom).

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