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Exogenous C/EBPα abrogates miR-182 mediated effects. a MiR-182 levels in <t>32D</t> cells transduced with either miR-182 or scramble lentivirus. MiR-182 expression was measured by qPCR. b MiR-182 expressing 32D cells showed increased cell numbers under G-CSF conditions ( black line ) compared to control scramble transduced cells ( gray line ). 1 × 10 5 cells were initially seeded and cultured in medium without IL-3 and supplemented with 20 ng/ml G-CSF. Data represent the mean ± SD of three independent experiments. (* p < 0.05; ** p < 0.01) P values were calculated using unpaired Student’s t -test. c Morphological analyses by cytospins followed by Wright-Giemsa-staining ( top panel ). MiR-182 expressing 32D cells exhibited less morphological signs of mature granulocytes and remained in an immature stage after treatment with G-CSF for 7 days. Representative plate from a CFU-assay of 32D-scramble or miR-182 expressing cells after 7 days under G-CSF conditions ( bottom panel ). Scale bar 5 µM. d Summary of 4 independent CFU-assays demonstrated enhanced colony forming unit ability of miR-182 expressing 32D cells after 7 days under G-CSF conditions. Data represent the mean ± SD. (** p < 0.01) P -values were calculated using unpaired Student’s t -test. e and f Stable overexpression of miR-182 led to a reduced percentage of Mac-1 positive 32D cells under both IL-3 and G-CSF conditions. e Representative flow cytometry analysis for myeloid marker Mac-1 in miR-182 or scramble expressing 32D cells under IL-3 conditions ( left panel ) or after treatment with 20 ng/ml G-CSF for 7 days ( right panel ) and f summary of all experimental repetitions. Y -axis indicate the percentage of Mac-1 positive cells. Data represent the mean ± SD of three independent experiments. (* p < 0.05; ** p < 0.01; *** p < 0.001) P -values were calculated using unpaired Student’s t -test. g Western blot analysis of 32D cells stably expressing either scramble or miR-182 sequence revealed reduced C/EBPα protein levels under IL-3 conditions or after treatment for 7 days with 20 ng/ml G-CSF. h Transduction of 32D cells with exogenous murine CEBPA enhances the detectable amount of C/EBPα protein. Analysis was performed using western blot. i Exogenous C/EBPα lacking a natural 3′UTR can rescue the miR-182 induced phenotype in 32D cells. Representative flow cytometry analysis for Mac-1 in either parental 32D cells or 32D cells stably expressing miR-182 transduced with control or m CEBPA . Analysis was made 10 days after transduction
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Exogenous C/EBPα abrogates miR-182 mediated effects. a MiR-182 levels in 32D cells transduced with either miR-182 or scramble lentivirus. MiR-182 expression was measured by qPCR. b MiR-182 expressing 32D cells showed increased cell numbers under G-CSF conditions ( black line ) compared to control scramble transduced cells ( gray line ). 1 × 10 5 cells were initially seeded and cultured in medium without IL-3 and supplemented with 20 ng/ml G-CSF. Data represent the mean ± SD of three independent experiments. (* p < 0.05; ** p < 0.01) P values were calculated using unpaired Student’s t -test. c Morphological analyses by cytospins followed by Wright-Giemsa-staining ( top panel ). MiR-182 expressing 32D cells exhibited less morphological signs of mature granulocytes and remained in an immature stage after treatment with G-CSF for 7 days. Representative plate from a CFU-assay of 32D-scramble or miR-182 expressing cells after 7 days under G-CSF conditions ( bottom panel ). Scale bar 5 µM. d Summary of 4 independent CFU-assays demonstrated enhanced colony forming unit ability of miR-182 expressing 32D cells after 7 days under G-CSF conditions. Data represent the mean ± SD. (** p < 0.01) P -values were calculated using unpaired Student’s t -test. e and f Stable overexpression of miR-182 led to a reduced percentage of Mac-1 positive 32D cells under both IL-3 and G-CSF conditions. e Representative flow cytometry analysis for myeloid marker Mac-1 in miR-182 or scramble expressing 32D cells under IL-3 conditions ( left panel ) or after treatment with 20 ng/ml G-CSF for 7 days ( right panel ) and f summary of all experimental repetitions. Y -axis indicate the percentage of Mac-1 positive cells. Data represent the mean ± SD of three independent experiments. (* p < 0.05; ** p < 0.01; *** p < 0.001) P -values were calculated using unpaired Student’s t -test. g Western blot analysis of 32D cells stably expressing either scramble or miR-182 sequence revealed reduced C/EBPα protein levels under IL-3 conditions or after treatment for 7 days with 20 ng/ml G-CSF. h Transduction of 32D cells with exogenous murine CEBPA enhances the detectable amount of C/EBPα protein. Analysis was performed using western blot. i Exogenous C/EBPα lacking a natural 3′UTR can rescue the miR-182 induced phenotype in 32D cells. Representative flow cytometry analysis for Mac-1 in either parental 32D cells or 32D cells stably expressing miR-182 transduced with control or m CEBPA . Analysis was made 10 days after transduction

Journal: Nature Communications

Article Title: Disruption of the C/EBPα—miR-182 balance impairs granulocytic differentiation

doi: 10.1038/s41467-017-00032-6

Figure Lengend Snippet: Exogenous C/EBPα abrogates miR-182 mediated effects. a MiR-182 levels in 32D cells transduced with either miR-182 or scramble lentivirus. MiR-182 expression was measured by qPCR. b MiR-182 expressing 32D cells showed increased cell numbers under G-CSF conditions ( black line ) compared to control scramble transduced cells ( gray line ). 1 × 10 5 cells were initially seeded and cultured in medium without IL-3 and supplemented with 20 ng/ml G-CSF. Data represent the mean ± SD of three independent experiments. (* p < 0.05; ** p < 0.01) P values were calculated using unpaired Student’s t -test. c Morphological analyses by cytospins followed by Wright-Giemsa-staining ( top panel ). MiR-182 expressing 32D cells exhibited less morphological signs of mature granulocytes and remained in an immature stage after treatment with G-CSF for 7 days. Representative plate from a CFU-assay of 32D-scramble or miR-182 expressing cells after 7 days under G-CSF conditions ( bottom panel ). Scale bar 5 µM. d Summary of 4 independent CFU-assays demonstrated enhanced colony forming unit ability of miR-182 expressing 32D cells after 7 days under G-CSF conditions. Data represent the mean ± SD. (** p < 0.01) P -values were calculated using unpaired Student’s t -test. e and f Stable overexpression of miR-182 led to a reduced percentage of Mac-1 positive 32D cells under both IL-3 and G-CSF conditions. e Representative flow cytometry analysis for myeloid marker Mac-1 in miR-182 or scramble expressing 32D cells under IL-3 conditions ( left panel ) or after treatment with 20 ng/ml G-CSF for 7 days ( right panel ) and f summary of all experimental repetitions. Y -axis indicate the percentage of Mac-1 positive cells. Data represent the mean ± SD of three independent experiments. (* p < 0.05; ** p < 0.01; *** p < 0.001) P -values were calculated using unpaired Student’s t -test. g Western blot analysis of 32D cells stably expressing either scramble or miR-182 sequence revealed reduced C/EBPα protein levels under IL-3 conditions or after treatment for 7 days with 20 ng/ml G-CSF. h Transduction of 32D cells with exogenous murine CEBPA enhances the detectable amount of C/EBPα protein. Analysis was performed using western blot. i Exogenous C/EBPα lacking a natural 3′UTR can rescue the miR-182 induced phenotype in 32D cells. Representative flow cytometry analysis for Mac-1 in either parental 32D cells or 32D cells stably expressing miR-182 transduced with control or m CEBPA . Analysis was made 10 days after transduction

Article Snippet: 32D (DSMZ) cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 10% WEHI-supernatant and 1% PSG.

Techniques: Transduction, Expressing, Control, Cell Culture, Staining, Colony-forming Unit Assay, Over Expression, Flow Cytometry, Marker, Western Blot, Stable Transfection, Sequencing