Fig 2: SERBP1-silencing downregulates Ago2 protein levels in SH-SY5Y cells. (A) Representative Western blot of extracts from cells transfected with control siRNA, Ago2 siRNA or SERBP1 siRNA from three independent experiments. Nitrocellulose membranes were hybridized with Ago2 or SERBP1 antibodies, as indicated. The GAPDH signal was used to normalize different samples (B). Fold change of Ago2 and SERBP1 protein levels in siRNA transfected cells relative to control siRNA samples are shown. Data represent the mean of three independent experiments ± standard error (S.E.); * p < 0.05 (t-test).. (C) RT-qPCR analysis of RNA extracted from SH-SY5Y cells transfected with control siRNA, Ago2 siRNA or SERBP1 siRNA. ?Ct values were normalized to TBP level. Fold change of Ago2 and Serbp1 mRNA expression levels relative to control siRNA samples are shown. Individual data points for Ago2 (circles) and Serbp1 (triangles) siRNA transfected cells are indicated. Data represent the mean of three independent experiments ± S.E.; * p < 0.05, ** p < 0.01.

Fig 3: SERBP1 or Ago2 silencing increases KCC2 expression. (A) Forty-eight hours after transfection with either control siRNA, Ago2 siRNA or SERBP1 siRNA (20 nM), SH-SY5Y cells were transfected with PSSG control plasmid or the Serpine1, or APP or KCC2 3'UTR firefly reporter plasmid. Firefly/renilla luminescence ratios were determined 24 h later. The data are presented as the fold increase of normalized luminescence ratios relative to cells transfected with control siRNA. Data represent the mean of three independent experiments +/- S.E.; * p < 0.05; ** p < 0.01. (B) Forty-eight hours after transfection with siAgo2 or siSERBP1 or a control siRNA (200 nM), hippocampal neurons were transfected with control plasmid or KCC2 3'UTR luciferase reporter. Firefly/renilla luminescence ratio was analyzed as described in (A). Data represent the mean of three independent experiments ± S.E. * p < 0.05. Individual data points for Ago2 (circles) and Serbp1 (triangles) siRNA transfected cells are indicated in (A) and (B) panels. (C) Representative Western blotting showing KCC2 upregulation in hippocampal neurons after SERBP1 silencing. Nitrocellulose membranes were hybridized with KCC2 or SERBP1 antibodies, as indicated. The GAPDH signal was used to normalize different samples. Means +/- S.E. of KCC2 and SERBP1 protein levels obtained from three independent experiments are presented relative to control cells. * p < 0.05; ** p < 0.01 (t-test).

Fig 4: SERBP1 interacts with KCC2 3'UTR. (A) SH-SY5Y cells were co-transfected with pCDNA3-myc or pCDNA3 myc-SERBP1 together with KCC2 3'UTR. Immunoprecipitates were analyzed by qRT-PCR to determine the levels of KCC2 mRNA in the two different conditions. Data represent the mean of three independent experiments ± S.E.; * p < 0.05. Individual data points for pcDNA3 myc-Serbp1 (squares) transfected cells are shown. (B) Western blotting showing Ago2 and SERBP1 protein levels in the input, eluates and supernatants from cytoplasmic lysates of SH-SY5Y cells (left panels) or hippocampal neurons (right panels) incubated with KCC2 sense or antisense 3'UTR transcripts. Eluate (1) and eluate (2) indicate the first and second elution. (C) Levels of miR-92 bound to KCC2 sense or antisense 3'UTR transcripts were analyzed by qRT-PCR. ?Ct values were normalized to the snU6 level *** p < 0.001.

Fig 5: SERBP1 silencing relieves miR-92a-mediated translational repression of KCC2 3'UTR reporter. (A) Forty-eight hours after transfection with control siRNA or SERBP1 siRNA (20 nM), SH-SY5Y cells were co-transfected with PSSG control plasmid or the reporter with the 3'UTR of KCC2 together with a control miRNA or miR-92a. Firefly/renilla luminescence ratios were determined 24 h later. The data are normalized to cells transfected with control siRNA and control miRNA. Data represent the mean of three independent experiments ± standard error. ns: not significant, * p < 0.05; ** p < 0.01 (t-test). Individual data points for siRNA SERBP1/miR-Ctr (triangles), siRNA Ctr/miR-92 (squares), siRNA Serbp1/miR-92 (rhombus) co-transfected cells are shown. (B) Representative Western blot showing the efficiency of SERBP1 silencing in SH-SY5Y used for luciferase assay. Means +/- S.E. of SERBP1 protein levels obtained from three independent experiments are presented relative to control cells. * p < 0.05.