Fig 1: Cochlear HC loss in neonatal mice up-regulates Lin28a and Lin28b expression.(A) Experimental scheme. To ablate HCs, Pou4f3DTR/+ mice and nontransgenic control littermates received DT (6.25 ng/g) at stage P1. To permanently label Deiter’s cells, pillar cells, and outer HCs, Fgfr3iCreERT2;R26tdTomato/+ tg mice received 4-hydroxytamoxifen (TM) injection at P0 in (F). (B to D) RT-PCR–based analysis of the expression of LIN28/let-7 (Lin28a, Lin28b, Hmga2, and Sox2) and TGF-β pathway (Fst, Bmp4, Inhba, and Tgfb2) genes in Ctrl (blue) and Pou4f3DTR/+ (DTR, red) cochlear sensory epithelia 24 (B), 48 (C), and 96 (D) hpd injection. (E) HMGA2 (red) expression in the HC-damaged (DTR) and undamaged Ctrl cochlear sensory epithelia in the apex, mid, and base 72 hours after DT injection. SC and HC layers are shown for undamaged control tissue. Lfng-GFP reporter expression marks SCs (green). (F) High-power images of tdTomato (magenta), HMGA2 (gray) and Atoh1-nGFP (green) expression in control and Pou4f3DTR/+ tg sensory epithelia 72 hpd injection. (G) Quantification of bright HMGA2+ cells within the cochlear sensory domain in (E) and (F). Two-tailed, unpaired t test was used to calculate P values in (B) to (D) and (G). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
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.
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