100 μm. Axon growth cones. Tubulin (red) + scr (green)
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1 Supplementary Figures mirorna-9 regulates axon extension an ranhing y targeting Map1 in mouse ortial neurons Dajas-Bailaor, F., Bonev, B., Garez, P., Stanley, P., Guillemot, F., Papalopulu, N. a 2 μm 1 μm p Relative RNA expression 15 p iz 1 5 U6 Tuulin (re) + mir-1 (green) e Tuulin (protein) Tuulin (re) + sr (green) f Tuulin (protein) mir-9 mir-13 mir-1 RNA Sr RNA Axon growth ones A. Tuulin (re)/mir-9 (green) Supplementary Figure 1. FISH of mir-9 on mouse rain setions at E17, showing expression in the (a) progenitor layer of the thir ventrile an () iz: intermeiate zone + white matter; p: ortial plate () RT-PCR analysis of mir-9 an mir-13 levels, emonstrating the relatively high expression of mir-9 in ortial neurons in ulture. Data are shown as perent of U6 RNA expression (mean + SEM). (-e) FISH of mir-1 an sramle (Sr) proes together with tuulin protein immunolaelling. Neither mir-1 nor Sr proes show loalisation in neuronal projetions (f) FISH of mir-9 together with aetylate tuulin protein immunolaelling in the axon growth ones of ortial neurons. Nature Neurosiene: oi:1.138/nn.382
2 Cell numer (% of total) Sramle p 1 p 2 p 3 p 4 p > 5 p s GFP + sramle GFP + mir-9 (5 nm) ax s ax GFP + LNA mir-9i (5 nm) ax s Supplementary Figure 2. (a) Distriution of the numer of neurons with to >5 ranh points in ontrol vs sramle mir proe transfete groups. Data are expresse as mean + SEM. () Representative images of neurons transfete with egfp plus sramle mir proe, mir-9 preursor or LNA mir-9i. s: enrites; ax: axons. Nature Neurosiene: oi:1.138/nn.382
3 Relative Lu. Ativity (%) WT mut Lu-Map1 3 UTR GFP-Map1 3 UTR-WT ontrol mir-9 GFP (% of ontrol) Cell oy WT mut WT mut GFP-Map1 3 UTR GFP-Map1 3 UTR-mut 5 μm 5 μm GFP + MAP1B GFP MAP1B (protein) 12 mir-9 sramle 1 μm 1 μm 1 μm MAP1B (% of ontrol) μm 1 μm 1 μm sr mir-9 5 nm e OD (%) ontrol mir-9 25 nm mir-9 5 nm sramle LNA mir-9i MAP1B GAPDH f Relative Map1 RNA levels ontrol sr mir-9 5 nm mir-9 ontrol 1 nm LNA LNA mir-9i 5 nm Supplementary Figure 3. Map1 is a target for mir-9 in ortial neurons. (a) Expression of luiferase reporter (Lu-Map1 3 UTR), wil type (WT) or with mutate (mut) mir-9 ining site, together with ontrol or mir-9 preursor () Loalize levels of expression of the same WT an mutate onstruts ut tagge to GFP an overexpresse in ortial neurons. () Representative images of ortial axons expressing GFP-Map1 3 UTR-WT (left) or GFP-Map1 3 UTR-mut (right). All images were proesse in parallel with iential aquisition settings. () Immunofluoresent etetion an quantifiation of enogenous MAP1B protein (re) in GFP positive ells, o-transfete with either sramle or mir-9 preursor in ortial neurons. Arrowheas iniate the axon of a transfete neuron, note ownregulation of MAP1B signal when mir-9 preursor is transfete. (e) Western lot of enogenous MAP1B or GAPDH proteins from ortial neurons after transfetion with mir-9 preursor or mir-9 LNA inhiitor. (f) RT-PCR of Map1 RNA levels in ortial neurons after transfetion with mir-9 preursor an LNA mir-9 inhiitor. Data is shown as fol hange with respet to ontrols. For all graphs ata are expresse as mean + SEM. Statistial signifiane ompare to respetive ontrols was etermine with Stuent s t test ( p <.5). Nature Neurosiene: oi:1.138/nn.382
4 Relative mir-9 levels (FC of ontrol) Ctrl LNA LNA mir-9i 5 nm Map1-TP 5 nm Relative mir-9 levels (FC of ontrol) Sr nm 1 nm 3 nm mir-9 Relative luiferase expression (FC of ontrol) LNA mir-9i (5 nm) Map1-TP (5 nm) Lu-Map1 3 UTR Lu-Hes1 3 UTR Lu-TLX 3 UTR Lu-Oneut1 3 UTR Supplementary Figure 4. Relative mir-9 levels after transfetion with the (a) LNA mir-9i an Map1- TP or with () mir-9 preursor in 17.2 ells. Data was normalize to snrna U6 an expresse as fol hange with respet to the ontrol. Inhiition of mir-9 ereases mature mir-9 levels, while the Map1-TP has no effet. () Relative luiferase expression levels in ifferentiate 17.2 ells o-transfete with either LNA mir-9i or Map1-TP an respetive luiferase reporters (Map1, Hes1, TLX, Oneut1). Inhiition of mir-9 inreases the expression levels of all targets, while the Map1-TP only has an effet on the Map1 3 UTR reporter. Data was expresse as fol hange ompare to the ontrol. For all graphs ata are shown as mean + SEM. Statistial signifiane ompare to respetive ontrols was etermine with Stuent s t test ( p <.5; p <.1). Nature Neurosiene: oi:1.138/nn.382
5 Somal sie Axonal sie um Axon length (% of ontrol) Axon length (% of ontrol) μm.1 μm.5 μm PNA mir-9i Map1 Relative Luiferase Ativity (FC of ontrol) LNA mir-9i 5 nm PNA mir-9i.1 μm (ell oy sie) PNA mir-9i.1 μm Supplementary Figure 5. (a) Shemati representation an representative image of a mirofluii hamer evie with its fluiially isolate axonal an somal ompartments (re: axonal aetylate tuulin, green: somal otx-1). () Measurement of axonal length on the axon sie after inuation with the PNA mir-9i only in the somati sie of the evie. No effet is oserve. () Measurement of axonal length in normal ortial ultures after inuation with the PNA mir-9i. Data for axonal length is shown as mean + SEM. Statistial signifiane was etermine using Mann-Whitney Rank Sum test for omparison with respetive ontrols. (p <.5). () Inrease in relative Lu-Map1 3 UTR ativity in 17.2 ells following inuation with LNA mir-9i or PNA-miR9-i. Data are expresse as fol hange to ontrols (mean + SEM). Statistial signifiane was etermine using Stuent s t test for omparison with respetive ontrols (p <.5; p <.1). Nature Neurosiene: oi:1.138/nn.382
6 mir-9 ensity (% of ontrol) BDNF: Axon sie axon length (% of ontrol) mir-9 ensity (% of ontrol) BDNF: Cell oy sie axon length (% of ontrol) ng ml-1 1 ng ml-1 1 ng ml-1 1 ng ml-1 Supplementary Figure 6. (a-) Quantifiation of mir-9 ensity an axonal length as measure on the axonal sie of the mirofluii evie with BDNF applie only to the (a-) axon or (-) ell oy sie of the hamers. Data is shown as mean + SEM. For axonal length experiments statistial signifiane was etermine using Mann-Whitney Rank Sum test for omparison with respetive ontrols. (p <.5). For mir-9 ensity omparisons, statistial signifiane was etermine using Stuent s t test for omparison with respetive ontrols. (p <.5; p <.1). Nature Neurosiene: oi:1.138/nn.382
7 A. Tuulin (re)/gw182 (green)/dapi (lue) a 1 μm Cell oy GW-182 fluoresene (% of ontrol) ng ml-1 mir9 12 mir-9 ensity (% of ontrol) ng ml-1 Supplementary Figure 7. (a) Protein loalization of the RISC omplex omponent GW-182 to the ell oy an istal axon in ortial neurons. (-) Quantifiation of GW-182 an omplementary mir-9 stran fluoresene in the istal axon after stimulation with BDNF. No statistially signifiant hanges were oserve, iniating that the effet of BDNF is not ue to a non-speifi moifiation of mirna levels or mirna proessing mahinery. Data is shown as mean + SEM. Nature Neurosiene: oi:1.138/nn.382
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