Fig 2: Reproductive stage dependent co-localization of Lin28 and DAZL in stallion germ cells.Three types of staining patterns such as Lin28 only, DAZL only, both Lin28 and DAZL were observed in both reproductive stages (k, l, m, n, and o). Lin 28 only positive germ cells and DAZL only positive germ cells were observed in post-pubertal stage (m and n). Germ cells labeled with both Lin28 and DAZL was also observed (o). No immunolabeling of Lin28 or DAZL was shown on tissues treated with rabbit igG instead of primary antibodies (p, q, r, and s). The white arrow head indicates germ cells stained with Lin28 only. The yellow arrow head indicates a germ cell stained with DAZL only. The blue arrow head indicates a germ cell which was co-immunolabeled with Lin28 and DAZL. Size bar = 200μm.

Fig 3: Expression of Lin28a and Lin28b in the whole embryo and mouse lung(A) qRT-PCR was conducted on two independent cohorts of the intestine, stomach, liver, heart, and brain from ages E12.5 to E18.5 (minus E16.5) and on three cohorts of lungs from ages E11.5 to adult by using DNA standards designed to amplify exon 2 for Lin28a and Lin28b. Error bars represent technical replicates of two independant biological samples for intestine, staomach, liver, heart, brain. Error bars represent biological replicates of three independant biological samples for lung(B) Immunoblots for protein expression were done with one of three cohorts of lung mentioned above from ages E12.5 to E18.5 by using mESCs as the control, conducted for Lin28a, Lin28b, and tubulin.(C) Immunofluorescence was conducted on an E11.5 wild-type (WT) frozen lung section for Lin28a, MynN, and DAPI.(D) We conducted qRT-PCR for Lin28a and Lin28b by using standards that amplify exon 2 of Lin28a and Lin28b from sorted lung fractions, n = 3 at E12.5. Error bars represent biological replicates of three independant biological samples. Scale bar, 0.5 mm.

Fig 4: The images of immunofluorescence staining of the E-cadherin, β-catenin and Lin 28 proteins in the mouse lungs at different phases of injury after irradiation. In the representative photomicrographs, the color for the E-cadherin (A) and Lin28 (C) proteins is red, the β-catenin protein (B) is green, and the DAPI-stained nuclei are blue. Magnification: 400x. h, hour; w, week; m, month.

Fig 5: Gene expression of the E-cadherin/β-catenin/Lin28 network detected by qRT-PCR in thoracically irradiated lungs at different phases of injury post-irradiation. The relative expression levels were calculated using the 2-△△CT method, and β-actin was used as the internal control. All the samples were analyzed in triplicate. *P < 0.05.