SUPPLEMENTARY FIGURE LEGENDS
Supplementary Fig. 1. Morphological analysis and quantification of differentiation markers
during in vitro neuronal differentiation of BE(2)-C cells. (A) Phase-contrast micrographs showing
morphological changes of BE(2)-C cells upon 3 or 6 days of RA treatment, compared to
unstimulated (-RA) cells. Elongation of neuritic processes and formation of cell aggregates are
visible. (B) qRT-PCR analysis showing the upregulation of neuronal differentiation markers (Vgf,
Tubb3, miR-124), and the parallel downregulation of the neural repressor Id2 and the proproliferative factor N-Myc in BE(2)-C cells treated with RA for different time points (days,
indicated below each bar). Histograms show mean values ± SD from three technical replicates.
Supplementary Fig. 2. Profiling of miR-125b-1 containing transcripts. (A) Schematic
representation of RefSeq MIR100HG and Non RefSeq MIR100HG/linc-NeD125. Exons are
represented as black (RefSeq MIR100HG) or grey (linc-NeD125) boxes. Arrowheads indicate
primer couples used for qRT-PCR analysis. (B) Histogram showing RefSeq MIR100HG and lincNeD125 quantities relative to Gapdh mRNA in BE(2)-C cells treated with RA for different time
points (days, indicated below each bar). Bars are colored according to the primers used, as in
panel (A). Error bars represent the standard deviation obtained from three technical replicates.
(C) Upper part: lncRNA_N2 genomic organization according to UCSC Genome Browser (assembly
Mar. 2006, NCBI 36/hg18). Lower part: histogram showing lncRNA_N2 (white bars) and lincNeD125 (grey bars) quantities relative to Gapdh mRNA in BE(2)-C cells treated with RA for
different time points. Primers used for lncRNA_N2 are the same as in Ng et al., 2012 29. Error bars
as in panel (B). (D) RT-PCR analysis of RefSeq MIR100HG, Non RefSeq MIR100HG/linc-NeD125,
BC089451 and AK123947 transcripts in the presence of oligo-dT or random hexamers from
untreated (day 0) or 6 days RA-treated BE(2)-C cells. Control reactions lacking Reverse
Transcriptase are loaded in lane –RT. Gapdh mRNA level was used as a loading control.
Supplementary Fig. 3. In silico and in vitro translation of linc-NeD125. (A) Prediction of small ORF
(10-100 aminoacids) occurrence from linc-NeD125 sequence using sORFfinder web-based tool.
(B) In vitro translation of linc-NeD125. Acrylamide fractionation of luciferase (as a control, lane
Luc) and linc-NeD125 (lane linc-NeD125) translation products. No protein products are
detectable either on long- or short-run gels (left and right panels, respectively). Molecular weight
marker is fractionated in lane M.
Supplementary Fig. 4. Expression profile of RefSeq MIR100HG and linc-NeD125 in D283 Med
cells. RefSeq MIR100HG (black bars) and linc-NeD125 (grey bars) quantities relative to Gapdh
mRNA in D283 Med cells during RA treatment for different time points (days). Error bars
represent the standard deviation obtained from three technical replicates.
Supplementary Fig. 5. Analysis of transcription factor levels and their association with lincNeD125 core promoter. (A) Analysis of REST, EGR1 and EGR2 protein levels during in vitro
neuronal differentiation. Left panel: Western blot analysis of protein extracts from BE(2)-C cells
treated with RA for the indicated times. GAPDH was used as a loading control. Right panel:
Western blot densitometric analyses. Results are normalized against GAPDH and expressed
relative to untreated cells (time 0, set as 1). Data are presented as mean values ± SEM from at
least three independent experiments. *p <0.05; **p <0.01; ***p <0.001. (B) Analysis of REST
occupancy on linc-NeD125 promoter. ChIP assay was performed in proliferating BE(2)-C cells,
immunoprecipitated with an antibody against REST or IgG. The region including the putative REST
binding site (-500) was PCR amplified. tRNAGlu was used as a negative control. (C) Analysis of
EGR1 association on linc-NeD125 promoter. Proliferating (-RA) or 6-days RA-treated (+RA) BE(2)C cells were immunoprecipitated with anti-EGR1 antibody or IgG. The -80 region, encompassing
the putative binding sites for the EGR factors, was PCR amplified, and tRNAGlu was used as a
negative control.
Supplementary Fig. 6. Putative binding sites for FUS, TDP-43 and HuR on linc-NeD125 sequence.
Supplementary Fig. 7. Efficiency and specificity of linc-NeD125 pull-down assay, and TDP-43 CLIP
and RNAi experiments. (A) RT-PCR analysis showing the specificity of linc-NeD125 recovery upon
RNA pull-down in differentiating BE(2)-C cells. Total extract was incubated with an antisense bait
specific for linc-NeD125 (α-NeD) or with a control bait (Ctrl). Primers used for RT-PCR allow the
simultaneous amplification of both linc-NeD125 isoforms in the Input RNA (lane IN). According
to the specificity of the α-NeD bait, a single band corresponding to the endogenous full-length
linc-NeD125 is visualized in lane α-NeD. Gapdh mRNA was used as a negative control. (B) Western
blot analysis assessing the efficiency of TDP-43 immunoprecipitation after CLIP assays in
proliferating (-RA) and differentiating (+RA) BE(2)-C cells. Total cell extracts were incubated with
anti-TDP-43 (lane TDP-43) or IgG (lane IgG) antibodies. Input sample (lane IN) was loaded as
control. (C) Western blot analysis of TDP-43 in proliferating (-RA) and differentiating (+RA) BE(2)C cells transfected with siRNAs against TDP-43 (lane siTDP-43), or with control siRNAs (lane siCtrl).
Supplementary Fig. 8. Expression profiles of Bcl2 and Bax in RA-treated BE(2)-C cells. qRT-PCR
analysis of Bcl-2 (left panel) and Bax (right panel) mRNAs during in vitro BE(2)-C differentiation.
Time points of RA treatment are indicated below. Histograms show mean values ± SD from three
technical replicates.
Supplementary Fig. 9. Analysis of differentiation markers upon modulation of linc-NeD125
expression. Neuronal differentiation markers Vgf, Tubb3, miR-124 and proliferative markers Id2
and N-Myc were analysed in (A) untreated (-Dox) or 3 days doxycycline-treated (+Dox) BE(2)-Clinc-NeD125 cells (or BE(2)-C-GFP cells as a control) and in (B) 3 days RA-treated BE(2)-C cells
transfected with esiRNA against linc-NeD125 (or against GFP, as a control). –Dox samples and
esiGFP were set as 1 in panel (A) and (B), respectively. Gapdh mRNA was used as a loading control.
Supplementary Table 1. List of oligonucleotide sequences used in this study.