Fig 2: c-Myc downregulates miR-23b and miR-27b expression in cultured neurons at the transcriptional level.A, Western blot analysis of c-Myc in primary cortical neurons 48 h after transfection with plasmids (pCMV6 or pCMV6-c-Myc). Relative amounts of the c-Myc protein were quantified by densitometry (n = 4, unpaired t-test, ** P<0.01). B, Quantitative RT-PCR detection of the expression of mature and primary form of miR-23b and miR-27b in primary cortical neurons pre-transfected with plasmids (n = 4, unpaired t-test, ** P < 0.01, and *** P<0.001). C, Western blot analysis of c-Myc in primary cortical neurons 48 h after transfection with c-Myc siRNA. Relative amounts of c-Myc protein were quantified by densitometry (n = 3, unpaired t-test, *** P<0.001). D, Quantitative RT-PCR detection of the expression of mature and primary form of miR-23b and miR-27b in primary cortical neurons pre-transfected with siRNA (n = 4, unpaired t-test, * P < 0.05).

Fig 3: Knockdown of c-Myc recovers the miR-23b and miR-27b levels decreased by hypoxia.A, Representative western blot images for c-Myc in the primary cortical neurons pre-transfected with control or c-Myc siRNAs under conditions of normoxia or hypoxia for 6 h. The relative amounts of the c-Myc protein were quantified by densitometry (n = 3, one-way ANOVA with Newman-Keuls multiple comparison test, ns, not significant, *** P<0.001). B, Quantitative RT-PCR detection of the expression of mature and primary form of miR-23b and miR-27b in primary cortical neurons pre-transfected with siRNA under conditions of normoxia or hypoxia for 6 h (n = 5, one-way ANOVA with Newman-Keuls multiple comparison test, ns, not significant, *** P<0.001). C and D, Representative western blot images for Apaf-1 (C) and quantitative RT-PCR detection of Apaf-1 mRNA expression (D) in the primary cortical neurons pre-transfected with control or c-Myc siRNAs under conditions of normoxia or hypoxia for 24 h. Relative amounts of Apaf-1 protein were quantified by densitometry (n = 3, one-way ANOVA with Newman-Keuls multiple comparison test, ns, not significant, *** P<0.001).

Fig 4: Small RNA loading, sub-cellular localization and polysome association of Tg-AGO.(A) Northern blot analyses of Tg-miRNAs and Tg -rdsRNAs associated with Tg-AGO. Immuno-precipitation using anti-Flag antibody was performed from RH strains expressing ectopically HAFlag-tagged either full-length or delta-RGG truncated Tg-AGO. RNAs isolated from the immunoprecipitates were probed for Tg-miR-4, Tg-miR-43, Tg-rdsRNA-17 and Tg-rdsRNA-28. Mock: non-immune IgG (negative control). (B) Stably expressed recombinant protein HAFlag-TgAGOFL was detected by immunofluorescence assay using an HA antibody (in green) and compared to nuclear localization of acetylated histone H4 (in red). (C) To evaluate the sedimentation characteristics of Tg-AGO complexes, protein extracts containing HAFlag-TgAGOFL or HAFlag-TgAGODRGG were subjected to sedimentation on 5%–45% sucrose gradients in the presence of cycloheximide (to preserve polyribosomes) or 30 mM EDTA (to disrupt polyribosomes). Aliquots of total extracts, either untreated (-RNase T1) or digested with RNase (+ RNase T1) were centrifuged through sucrose gradients and fractionated as described in Methods. Aliquots (equal volume) from indicated fractions were analyzed by Western blots with antibody against HA tag. (D) HAFlag-TgAGOFL and HAFlag-645.m00319 (negative control) were transiently co-expressed with Myc-Tg-p100 (Tudor/SN). Following Flag affinity purification, the bound proteins were analyzed by Western blot using the anti-HA and the anti-myc antibodies. Molecular weight markers are indicated on the left.

Fig 5: c-Myc acts on E-Boxes in the promoter of the miR-23b-27b cluster.A, Diagram representing the c-Myc binding sites in the miR-23b-27b cluster promoter region. B and C, ChIP assays were performed by IP with either anti-c-Myc antibody or control IgG. D, c-Myc regulates the promoter of the miR-23b-27b cluster. For the luciferase reporter assay, cells were co-transfected with empty vector or c-Myc plasmids and the luciferase reporter plasmid carrying promoter constructs containing site 1 or site 1 mutant (n = 3, unpaired t-test, ns, not significant,** P < 0.01).