Fig 2: IL-6 increases STAT3 transcript and protein in neurons and induces selective phosphorylation at Tyr-705(A) Experimental workflow: IL-6 at 10 ng/mL was added at 1 and 4 DIV, and neurons were collected at 6 DIV for the analysis.(B) qPCR analysis of Stat3 mRNA in ctrl and IL-6-treated cultures (4 independent experiments, one-sample t test, **p = 0.008).(C) Western blot analysis of STAT3 protein in ctrl and IL-6-treated neuronal cultures in the absence (with astrocytes, left panel) or presence of Ara-C (no astrocytes, right panel).(D) Quantitative analysis of the optical density of STAT3 immunoreactive bands normalized by GAPDH in untreated (left graphs) and Ara-C-treated cultures (right graphs). Four independent experiments; one-sample t test; Ctrl cultures, *p = 0.025; Ara-C cultures, *p = 0.041).(E) Experimental workflow: a single pulse of vehicle (saline, as a ctrl) or IL-6 was injected i.p. into pregnant mothers on GD15 and the Hp of the embryo was analyzed 24 h after injection through immunofluorescence.(F) Immunofluorescence of hippocampal regions stained with STAT3 (red) and Hoechst (blue), established from embryos on E16 exposed to vehicle or IL-6 on GD15 via maternal i.p. injection. VZ, ventricular zone; HNE, hippocampal neuroepithelium; DNE, dentate neuroepithelium; CH, cortical hem. Inset panels: higher magnification of the selected area. Scale bars, 250 µm.(G) Quantitative analysis of normalized STAT3 mean intensity in the Hp under the two conditions (vehicle, n = 7 embryos; IL-6, n = 6 embryos; Three independent experiments (3 litters); Student’s t test; *p = 0.0129).(H) Cultured neurons at 6 DIV were treated acutely with IL-6 and collected at different time points for western blot analysis.(I and J) Time course analysis of STAT3 phosphorylation at Tyr-705 (I) and Ser-727 (J) upon acute (30-min) IL-6 treatment at 6 DIV in cultured neurons. Values were normalized (bottom panels) against the total amount of STAT3 (4 independent experiments, Wilcoxon signed-rank test, *p = 0.0313).See also Figure S5.

Fig 3: PTX3 does not affect synapse number and organization ARepresentative images of 14DIV control and PTX3-treated cultures stained for the presynaptic marker Bassoon (blue), the postsynaptic protein PSD95 (green), and the microtubule protein tubulin (red). Scale bar: 5 µm.BQuantification of synaptic density shows no differences either for postsynaptic marker (PSD95/µm), or for presynaptic marker (Bsn/µm) or as a total number of synapses (PSD95&Bsn/µm) in control and PTX3-treated cultures (PSD95/µm, Ctr = 0.391 ± 0.018; PTX3 = 0.379 ± 0.016; Bsn/µm, Ctr = 0.291 ± 0.012, PTX3 = 0.311 ± 0.012; PSD95&Bsn/µm, Ctr = 0.218 ± 0.009, PTX3 = 0.235 ± 0.013. Number of dendrites: 109 Ctr, 107 PTX3, Mann–Whitney test; five independent experiments, data are presented as mean ± SEM).CQuantitative analysis of the mean size of PSD95 and Bsn puncta shows no differences in control or PTX3-treated neurons (in µm2, PSD95: Ctr = 0.155 ± 0.010; PTX3 = 0.158 ± 0.008; Bsn: Ctr = 0.205 ± 0.013, PTX3 = 0.214 ± 0.012; Number of dendrites: 134 ctr, 124 PTX3, Mann–Whitney test; five independent experiments, data are presented as mean ± SEM).D, E(D) Representative images of GFP-expressing dendritic branches of control and PTX3-treated neurons and (E) quantification of dendritic spine density, i.e., number of spines per µm of parent dendrite (Ctr = 0.607 ± 0.020, PTX3 = 0.598 ± 0.021, number of examined dendrites: 73 and 71 respectively; Mann–Whitney test; three independent experiments, data are presented as mean ± SEM). Scale bar: 5 µm.FWestern blotting analysis of major synaptic proteins on lysates from control or PTX3-treated cultures. GAPDH was used as reference marker.G–IChronic PTX3 administration does not increase neither synaptic density nor synaptic puncta size. (G) Representative images of 14DIV control and PTX3-treated cultures stained for the presynaptic marker Bassoon (blue), and the postsynaptic protein PSD95 (green) and tubulin (red). Scale bar: 5 µm. (H) Quantification of synaptic density parameters (PSD95/µm; Bsn/µm; PSD95&Bsn/µm) in control and PTX3-treated neurons (PSD95/µm, Ctr = 0.297 ± 0.029; PTX3 = 0.334 ± 0.026; Bsn/µm, Ctr = 0.261 ± 0.021, PTX3 = 0.221 ± 0.020; PSD95&Bsn/µm, Ctr = 0.209 ± 0.019, PTX3 = 0.148 ± 0.014. Number of dendrites: Ctr = 20, PTX3 = 40, Mann–Whitney test; three independent experiments; data represented as mean ± SEM). (I) Analysis of the mean size of PSD95 and Bsn puncta shows no differences in control or PTX3-treated neurons (in µm2, PSD95: Ctr = 0.081 ± 0.009; PTX3 = 0.094 ± 0.010; Bsn: Ctr = 0.175 ± 0.021, PTX3 = 0.138 ± 0.016; number of dendrites: Ctr = 22, PTX3 = 48, Mann–Whitney test; three independent experiments, data represented as mean ± SEM). Source data are available online for this figure.

Fig 4: The individual protein expression levels of NTM and NEGR1 in the frontal cortex of wild-type and Lsamp-/- mice measured by western blot. (A) NTM and (B) NEGR1 expression levels are presented relative to GAPDH; n = 7; *p < 0.05. It is important to note that NTM antibody is not able to discriminate between 1a and 1b isoforms, therefore the significant decrease of Ntm 1a in Lsamp-/- animals (Figure 4A) is only detectable in mRNA level.

Fig 5: Otoferlin dual-AAV-TS-transduced Otof -/- organs of Corti express full-length otoferlin mRNA ASchematic representation of otoferlin cDNA from otoferlin dual-AAV-transduced, wild-type, and Otof -/- organs of Corti, displaying binding sites of primers used in PCRs to assess dual-AAV reassembly.BOtoferlin PCR amplicons from organ of Corti cDNA. A 1,753-bp-long amplicon (*), also present in non-injected wild-type controls (WTB6, WTCD1B6F1), indicates successful reassembly of the split otoferlin expression cassette in otoferlin dual-AAV-TS-transduced CD1B6F1-Otof -/- organs of Corti (injected ear). In Otof -/- samples, three shorter products were amplified (a, b and c).CSanger sequencing confirmed correct dual-AAV split-site assembly (dashed line) as well as the presence of an artificial AccIII restriction site introduced in the dual-AAV-TS otoferlin cDNA, which is absent in the wild-type (WT) and Otof -/- cDNA (a–c). Amplicons a-c from Otof -/- organs of Corti all lack exons 14–15, while bands “b” (1,480 bp) and “c” (1,679 bp) still contain intron 20–21 (b) or intron 23–24 (c), respectively.DWestern blotting on cell lysates of WT and Otof -/- CD1B6F1 organs of Corti. Two bands of ˜210–230 kDa, corresponding to full-length otoferlin, were detected in WT but absent in Otof -/- ears. (**) refers to an unspecific band detected in both samples. GAPDH was used as loading control.Data information: CDS: coding sequence, Ex: exon, TS: trans-splicing, Hyb: hybrid, control ear: non-treated ears, non-injected ear: contralateral non-injected Otof -/- ears.Source data are available online for this figure.