Fig 1: Down-regulated MYC signaling in SOX1-induced quiescent NPC cells.A Western blot analysis of SOX1, p27Kip1 and β-actin expression in “Proliferation” and “Quiescence” group of cells. β-actin was used as a control. B GSEA of ranked genes from cells. Gene set “hsa03010: Ribosome” of KEGG pathways was shown. NES normalized enrichment score. C Western blot analysis of RPS3, RPL7A, c-MYC and β-actin expression in “Proliferation” and “Quiescence” group of cells. β-actin was used as a control. D Ridgeplots showing MSigDB Hallmark gene sets based on the GSEA analysis of proliferative and quiescent NPC cells. The “Proliferation” (blue) or “Quiescence” group (red) of cells was enriched in the indicated gene sets. E Motifs analysis of SOX1-binding peaks. The top de novo motif for SOX1-binding is presented, along with several similar known motifs. F Matched sequences to SOX1-binding de novo motif in MYC promoter. A higher score indicates that the match is more significant. G Upper panel, truncated designs of different MYC promoters on a luciferase reporter vector. Lower panel, dual luciferase assay evaluating the effects of SOX1 on the truncated (2k, 1.5k, 1k, and 0.5k) MYC promoter reporter constructs. H Upper panel, mutant designs of different MYC promoters on a luciferase reporter vector. Lower panel, dual luciferase assay evaluating the effects of SOX1 on the wild-type (WT) or mutant (MU1 and MU2) MYC promoter reporter constructs.
Fig 2: SOX1 decreases ribosome pathway and DNA replication in NPC cells.A An experimental timeline for doxycycline (Blue block: 0 µg/ml, red block: 1 µg/ml) schedules used to control the expression of SOX1 in cells (HONE1 TRE-SOX1 or CNE2 TRE-SOX1). On day 2 and day 4, the cells were counted and proteins were extracted for protein concentration and western blot analysis, respectively. B Western blot analysis of SOX1, RPS3, RPL7A, and β-actin expression in cells. β-actin was used as a control. C Gene set enrichment analysis (GSEA) of ranked genes from cells. Gene set “hsa03010: Ribosome” of KEGG pathways was presented. NES normalized enrichment score. D Scatter dot plots showing the average protein content per cell. E An experimental timeline for doxycycline (Blue block: 0 µg/ml, red block: 1 µg/ml) schedules used to control the expression of SOX1 in cells (HONE1 TRE-SOX1 or CNE2 TRE-SOX1). Both groups of cells were treated with 20 µM BrdU for varying durations, and on day 4, BrdU-positive cells were determined by flow cytometry analysis. F Histograms showing the percentage of BrdU-negative or -positive cells in “SOX1-High” and “SOX1-Low” groups of cells. G Time courses of the percentage of BrdU-positive cells in BrdU incorporation assays, which were fitted by an exponential plateau equation. H An illustration of model hypothesis that overexpression of SOX1 induces quiescent NPC cells. Dox Doxycycline.
Fig 3: Single-cell sequencing analysis of different variants of SARS-CoV-2. (A) Annotation of viral particles in 16HBE cells infected with the Wuhan (left) and XBB (right) strains. (B) Detection of E-gRNA and E-sgRNA in 16HBE cells infected with the Wuhan and XBB strains via Q-PCR. (C) Differential pathway enrichment in 16HBE cells infected with the Wuhan or XBB strains via pseudotime analysis. (D) Comparison of host differentially expressed gene pathway enrichment in 16HBE cells infected with the Wuhan or XBB strain, GO enrichment analysis. (E) Comparison of the protein expression levels of the ribosome related factors RPS3 and eIF4E between the Wuhan strain and the XBB strain via Western blotting. (F) Comparison of the expression levels of RPS3 in 16HBE cells infected with the Wuhan or XBB strain. **** P < 0.0001.
Fig 4: Schematic illustration of the mechanism of LXN/Rps3/p53 pathway on cellular senescence in CaOx stone formation. Activation of LXN gene in renal tubular epithelial cells (RTECs) is evoked by hyperoxaluria and oxalate. Since ribosomal protein subunit 3 (Rps3) is a LXN binding protein, the nuclear translocation of Rps3 were significantly down-regulated due to the more combination conducted by LXN protein. And then p53 which might be a downstream target of Rps3 is up-regulated, with a consequence of triggering RTECs’ senescence. Senescence-Associated Secretory Phenotype (SASP) factors are then released, skewing macrophage polarization towards pro-inflammatory M1 phenotype, not anti-inflammatory M2 phenotype, demonstrating a low crystals phagocytic ability. Therefore LXN gene targeting silencing could effectively inhibit cellular senescence, thus reduce macrophage polarization towards M1 phenotype and ultimately behave a protective effect on kidney stone formation.
Fig 5: LXN/Rps3/p53 pathway was involved in the oxalate induced senescent cell burden. (A) Laser confocal microscopy was applied to evaluate nuclear localization of Rps3. Nuclear localization of Rps3 were significantly decreased in HK-2 cells under oxalate induction compared to control cells, and these decreased nuclear translocation of Rps3 were significantly up-regulated by fisetin treatment. mRNA (B) and protein (C) expression of Rps3 in HK-2 cells under oxalate with/without fisetin treatment appeared no significant changes compared to control group. Oxalate induction was performed on the basis of LXN knockdown. (D) LXN knockdown has little effect on expression levels of Rps3/p53 mRNA, and Rps3 silencing has little effect on LXN3/p53 mRNA expression. (E) p53 protein expression was significantly down-/up-regulated by LXN/Rps3 knockdown. (F) Nuclear translocation of Rps3 was obviously elevated after LXN silencing. Data are presented as means ± SD. **P < 0.01, ***P < 0.001; CTL, control group. Scr siRNA=Scrambled siRNA control.
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