Figure 2.
Figure 2. Molecular characterization of FNDC3B-RARA and implications of FNDC3B in granulocytic differentiation. (A) Immunofluorescence analysis of FNDC3B-RARA and FNDC3B localization. HeLa cells were transfected with the indicated C-terminally hemagglutinin (HA)-tagged expression plasmids, and the tagged proteins were detected using an anti-HA antibody followed by an Alexa Fluor 488–conjugated secondary antibody. 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. PML-RARA exhibited a microspeckled nuclear pattern as expected. Original magnification ×1000. (B) Analysis of FNDC3B-RARA homodimerization. HeLa cells were transfected with the indicated combinations of C-terminally Myc-tagged and HA-tagged FNDC3B-RARA expression vectors. pCMV-Myc and pCMV-HA are empty vectors. Myc-tagged proteins were immunoprecipitated and samples were analyzed by immunoblotting using the indicated antibodies. (C) Analysis of FNDC3B-RARA heterodimerization with RXRA. Myc-tagged FNDC3B-RARA expression vector was cotransfected with pCI-RXRA or pCI-RARA into HeLa cells. Myc-tagged proteins were immunoprecipitated and samples were analyzed by immunoblotting. FNDC3B-RARA interacts with RXRA but not RARA. In panels B and C, FR indicates FNDC3B-RARA. Twenty micrograms of cell lysates were analyzed in all input samples. (D) Unliganded FNDC3B-RARA is a potent transcriptional repressor. The Cignal RARE reporter was cotransfected with pCI-RARA (RARA), pCI-FNDC3B-RARA (FR), pCI-PML-RARA (PR), or the empty pCI vector (EV) into HeLa and U937 cells. Luciferase activities were measured 30 hours after transfection. Results are presented as relative luciferase activity by comparing to the empty vector control. **P < .01 and ***P < .0001 vs RARA, respectively. (E) The transcriptional activity of FNDC3B-RARA is strongly stimulated by ATRA. After 24 hours of cotransfection with the RARE reporter and various pCI expression vectors as described in panel D, cells were treated with the indicated concentrations of ATRA or dimethyl sulfoxide (DMSO) (vehicle control) for 6 hours before luciferase measurement. Results are presented as fold activation by comparing ATRA treatment to DMSO. *P < .05 and **P < .01, respectively. In panels D and E, transfection efficiency was normalized based on the constitutively expressing Renilla luciferase construct in the RARE reporter and results are expressed as mean ± standard error (SE) from at least 3 independent experiments each performed in triplicate. (F) FNDC3B knockdown impaired ATRA-induced differentiation of NB4 cells. Top, Confirmation of FNDC3B knockdown after 48 hours of siRNA transfection by western blotting. β-actin served as the loading control. Twenty four hours after siRNA transfection, NB4 cells were treated with 100 nM ATRA or DMSO (vehicle control) for 3 days. CD11b expression was measured by flow cytometry (middle) and CCAAT/enhancer-binding protein ε (CEBPE) expression by quantitative RT-PCR and normalized to GAPDH (bottom). Results are expressed as mean ± SE from 3 independent experiments. For CEBPE, expression levels were relative to DMSO treatment. (G) FNDC3B knockdown enhanced NB4 cell proliferation. Twenty four hours after siRNA transfection, NB4 cells were subjected to WST-1 (top) and colony-forming assays (bottom) for cell proliferation analysis. Results are expressed as mean ± SE from 3 independent experiments. In panels F-G, *P < .05 and **P < .01 vs control siRNA, respectively. IP, immunoprecipitation; RFU, relative fluorescence unit.

Molecular characterization of FNDC3B-RARA and implications of FNDC3B in granulocytic differentiation. (A) Immunofluorescence analysis of FNDC3B-RARA and FNDC3B localization. HeLa cells were transfected with the indicated C-terminally hemagglutinin (HA)-tagged expression plasmids, and the tagged proteins were detected using an anti-HA antibody followed by an Alexa Fluor 488–conjugated secondary antibody. 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. PML-RARA exhibited a microspeckled nuclear pattern as expected. Original magnification ×1000. (B) Analysis of FNDC3B-RARA homodimerization. HeLa cells were transfected with the indicated combinations of C-terminally Myc-tagged and HA-tagged FNDC3B-RARA expression vectors. pCMV-Myc and pCMV-HA are empty vectors. Myc-tagged proteins were immunoprecipitated and samples were analyzed by immunoblotting using the indicated antibodies. (C) Analysis of FNDC3B-RARA heterodimerization with RXRA. Myc-tagged FNDC3B-RARA expression vector was cotransfected with pCI-RXRA or pCI-RARA into HeLa cells. Myc-tagged proteins were immunoprecipitated and samples were analyzed by immunoblotting. FNDC3B-RARA interacts with RXRA but not RARA. In panels B and C, FR indicates FNDC3B-RARA. Twenty micrograms of cell lysates were analyzed in all input samples. (D) Unliganded FNDC3B-RARA is a potent transcriptional repressor. The Cignal RARE reporter was cotransfected with pCI-RARA (RARA), pCI-FNDC3B-RARA (FR), pCI-PML-RARA (PR), or the empty pCI vector (EV) into HeLa and U937 cells. Luciferase activities were measured 30 hours after transfection. Results are presented as relative luciferase activity by comparing to the empty vector control. **P < .01 and ***P < .0001 vs RARA, respectively. (E) The transcriptional activity of FNDC3B-RARA is strongly stimulated by ATRA. After 24 hours of cotransfection with the RARE reporter and various pCI expression vectors as described in panel D, cells were treated with the indicated concentrations of ATRA or dimethyl sulfoxide (DMSO) (vehicle control) for 6 hours before luciferase measurement. Results are presented as fold activation by comparing ATRA treatment to DMSO. *P < .05 and **P < .01, respectively. In panels D and E, transfection efficiency was normalized based on the constitutively expressing Renilla luciferase construct in the RARE reporter and results are expressed as mean ± standard error (SE) from at least 3 independent experiments each performed in triplicate. (F) FNDC3B knockdown impaired ATRA-induced differentiation of NB4 cells. Top, Confirmation of FNDC3B knockdown after 48 hours of siRNA transfection by western blotting. β-actin served as the loading control. Twenty four hours after siRNA transfection, NB4 cells were treated with 100 nM ATRA or DMSO (vehicle control) for 3 days. CD11b expression was measured by flow cytometry (middle) and CCAAT/enhancer-binding protein ε (CEBPE) expression by quantitative RT-PCR and normalized to GAPDH (bottom). Results are expressed as mean ± SE from 3 independent experiments. For CEBPE, expression levels were relative to DMSO treatment. (G) FNDC3B knockdown enhanced NB4 cell proliferation. Twenty four hours after siRNA transfection, NB4 cells were subjected to WST-1 (top) and colony-forming assays (bottom) for cell proliferation analysis. Results are expressed as mean ± SE from 3 independent experiments. In panels F-G, *P < .05 and **P < .01 vs control siRNA, respectively. IP, immunoprecipitation; RFU, relative fluorescence unit.

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