Fig 1: Enhanced chondrogenesis in pellets of AF cells by knockdown of Pax1. (a) AF cells isolated from 4-week-old Wistar rats were seeded at a density of 8 × 104 cells/well in a 12-well plate. Morphology of AF cells cultured in DMEM containing 10% FBS at day 1 is shown. (b) Total RNA was extracted from AF cells maintained in a culture dish for 96 h and then processed for RT-PCR analysis of expression of ECM molecules (Acan, Col1a2, Col2a1, Ocn) and transcription factors (Pax1, Pax9, Nkx3.2, Sox5, Sox6, Sox9). Full-length gels are presented in Supplementary Fig. 7. (c) AF cells were seeded at a density of 4 × 104 cells/well in a 24-well plate. At 24 h after inoculation, the cells were transfected with non-targeting siRNA (control) or Pax1 siRNAs (siPax1-a or siPax1-b) by lipofection. Total RNA was extracted from the cells at 48 h after lipofection, and the expression levels of Pax1 or Acan were examined by qRT-PCR. (d) Experimental schedule of chondro-induction in pelleted cultures of AF cells. AF cells were seeded at a density of 1 × 105 cells/well in 6-well plates and then infected with shRNA lentiviruses on day 1. After trypsinization on day 3, cell pellets were prepared from 1.5 × 105 cells/pellet and cultured in chondrogenic differentiation medium for another 21 days. (e–g) Sections of cell pellets infected with shRNA lentiviruses were stained with alcian blue. Increased accumulation of alcian blue-stained extracellular matrix was observed in the pellets of AF cells infected with shPax1-a or shPax1-b lentivirus (f,g) compared to that in cells infected with control lentivirus (e). (h) Total RNA was extracted from pellets on day 24. Relative expression levels of Pax1, Sox9, and Acan were examined by qRT-PCR analysis. qRT-PCR data (c,h) represent the average of three independent experiments. The relative expression of each gene is normalized to that in the control and reported as mean ± s.d. ***P < 0.001 versus control. Scale bars in (a) and (e–g), 100 μm.
Fig 2: circ-LDLRAD3 knockdown suppressed tumor growth in vivo. (A) During the period of tumor growth, tumor volume was measured every 3 days. (B) Tumor weight was measured after tissue excision. (C) The expression of circ-LDLRAD3 and miR-588 in tumor tissues was detected by qPCR. (D) The expression of SOX5 protein in tumor tissues was measured by Western blotting. (E) The abundance of SOX5 and Ki67 in tumor tissues was monitored by immunohistochemistry assay (40×).circ-LDLRAD3, hsa_circ_0006988; qPCR, quantitative real-time polymerase chain reaction; SOX5, SRY-box transcription factor 5; PBS, phosphate-buffered saline; DDP, cisplatin; NC, negative control. *p<0.01, †p<0.001, ‡p<0.0001.
Fig 3: SOX5 is a direct target of miR-194-5p and its upregulation reverses the impacts of miR-194-5p on CRC cells.A The putative binding site between SOX5 and miR-194-5p through prediction of TargetScan online software. B luciferase reporter assay for analysis of the interaction between SOX5 and miR-194-5p, ***P < 0.001 vs. miR-NC. C western blotting was performed to detect the protein expression levels of SOX5 in CRC tissues and CRC cell lines. D qRT-PCR was performed to determine the effect of miR-194-5p-mimic in decreasing SOX5 expression in HT29 and SW480 cells, ***P < 0.001 vs. miR-NC. E HT29 and SW480 cells were transfected with pcDNA3.1-SOX5 and the fold changes in the expression of SOX5 was analyzed, ***P < 0.001 vs. pcDNA-Con; EDU (F), migration (G), and invasion (H) assays were conducted respectively in HT29 and SW480 cells mentioned above, ***P < 0.001 vs. miR-NC + pcDNA-Con; ##P < 0.01, ###P < 0.001 vs. miR-mimic + pcDNA-Con. Scale bar = 50 μm. Data were expressed as mean ± SD. All experiments were repeated three times.
Fig 4: CSSEDF-expanded cells stably self-renewed and shared markers of CPCs/CSCs.a A single CSSEDF-expanded cell (passage 5) colony on a Matrigel-coated surface. Scale bars, 50 µm. b–d Immunocytochemistry showing that CSSEDF-expanded cells expressed genes identified as neural ectodermal markers, including SIX1, NESTIN and ETS1. Scale bars, 50 µm. e–i Immunocytochemistry showing that CSSEDF-expanded cells (passage 5) expressed genes identified as CPC/CSC markers, including SOX9, RUNX2, SOX5, TWIST1, and CD29. Scale bars, 50 µm. j–l Immunocytochemistry showing that CSSEDF-expanded cells (passage 5) express genes recently identified as TMJ condylar cartilage markers, including FOXC1, FOXC2 and MSX1. Scale bars, 50 µm. m Flow cytometry analysis showing that CSSEDF-expanded cells stably express cell proliferation and CPC/CSC markers after long-term in vitro expansion. n Gene expression of chondrocytic lineage markers by CSSEDF-expanded cells at different passages and patient TMJ condylar cartilage were analysed by PCR. CPCs/CSCs: cartilaginous progenitor cells/cartilaginous stem cells.
Fig 5: Establishment of the typical fetal lamination during the mid-fetal period (15 PCW). Magnified regional portions of the Nissl coronal section show a cytoarchitectonic overview of the typical fetal lamination (A–D). IF-stained sections show regional and laminar dynamics of DPN markers expression (A.1–D.3). Expanded SP shows TBR1 reactivity throughout the whole thickness (A.2–D.2). SPN are labeled with deep-projection neuron markers TLE4 (A.2–D.2), SOX5, and CTIP2 (A.1–D.1), as well as CELF1 (A.3–D.3). Note that SOX5 shows the strongest signal in the most superficial part of the CP. CTIP2 shows a strong signal in the lower CP and the SP. TLE4 is expressed in the lower CP and SP. In addition, DCX positive signal marks migratory zones from VZ to CP. Layer IV and III neurons are already born during the mid-fetal period. ((A), (A.1–A.3)—lateral, (B), (B.1–B.3)—dorsal, (C), (C.1–C.3)—medial, and (D), (D.1–D.3)—basal portion of the frontal cortex). Scale bar = 200 μm.
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