Fig 2: TGF-β2 inhibits HC formation in LIN28B-overexpressing stage P5 cochlear organoids.Organoid cultures were established with cochlear epithelial cells from stage P5 control, iFST, iLIN28B, and iFST;iLIN28B tg mice. (A) Experimental scheme (B to D). DIV, day(s) in vitro. (B) Low-power bright-field images show that exogenous TGF-β2 inhibits organoid formation and growth. (C) Organoid diameter in (B) (n = 4, from one representative experiment and three independent experiments). (D) Organoid-forming efficiency in (B) (n = 4, from one representative experiment and three independent experiments). (E) Experimental scheme (F to I). (F) Low-power bright-field and green fluorescent (Atoh1-nGFP) images show that exogenous TGF-β2 blocks HC formation in FST + LIN28B–overexpressing cultures. (G) Percentage of Atoh1-nGFP+ organoids in LIN28B-overexpressing cultures treated with and without TGF-β2 (n = 4, from one representative experiment and three independent experiments). (H) Percentage of Atoh1-nGFP+ organoids in (F) (n = 3, untreated group; n = 4, TGF-β2–treated groups; from one representative experiment and three independent experiments). (I) RT-PCR analyzing Atoh1 (blue) and Pou4f3 (red) mRNA induction in (F) (n = 3, from one representative experiment and three independent experiments). (J) Experimental scheme (K to N). (K) RT-qPCR analyzing the Tgfb2 knockout efficiency using Tgfb2-shRNA lentiviral particles. WT, wild-type. (L) RT-PCR of HC-specific (Atoh1, Pou4f3, and Gfi1) mRNA expression (n = 3, from one representative experiment and two independent experiments). (M) Bright-field, mCherry (red), and green fluorescent (Atoh1-nGFP) images of LIN28B-overexpressing organoids infected with control (scr shRNA) or Tgfb2-shRNA–expressing lentivirus. (N) Percentage of Atoh1-nGFP+organoids in (M) (n = 3, from one representative experiment and two independent experiments). One-way ANOVA with Tukey’s correction was used to calculate P values in (C) to (H). Two-tailed, unpaired t test was used to calculate P values in (I) to (N). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 3: Developmental changes of Tgfβ expression and its effects on PNN and lectican levels.(A) The expression of Tgfb1 and Tgfb2 mRNA decreases in the RSP but not hippocampus between p30 and p75 (unpaired t test: RSP Tgfb1: n = 4, t6 = 4.723 p = 0.003; RSP Tgfb2: n = 4, t6 = 7.966, p < 0.001). (B) The methylation change between p75 and p30 on selected CpG islands of Tgfb2 gene, significant marks represent significant differences between p30 and p75 percentage of methylation on corresponding CpG islands (n = 4, Chr1:186438127: t6 = 2.756, p = 0.033, Chr1:186438103: t6 = 3.231, p = 0.018, Chr1:186438077: t6 = 2.466, p = 0.049, Chr1:186437896: t6 = 2.449, p = 0.050, Chr1:186407019: t6 = 3.209, p = 0.018, Chr1:186359949: t6 = 2.992, p = 0.024, Chr1:186359929: t6 = 5.222, p = 0.002, Chr1:186359904: t6 = 4.877, p = 0.003, Chr1:186359899: t6 = 4.129, p = 0.006). (C) Intra-RSP Tgfβ2 injection attenuates the loss of PNNs (n = 8; unpaired t test: t14 = 2.278, p = 0.039), Ncan (n = 8; unpaired t test: t14 = 4.325, p < 0.001), and PV (n = 8; unpaired t test: t14 = 3.937, p = 0.002) expressions one week later. Experimental diagrams were created in BioRender. Zhang, H. (2026) https://BioRender.com/tgo8isf. Data represent mean ± s.e.m., ***p < 0.001; **p < 0.01; *p < 0.05. The data underlying this Figure can be found in S1 Data.