Fig 1: RTN3 modulates the loading of infectious viral molecules inside exosomes.(A&B) Reticulon 3L&S (RTN3L&S) was knockdown using specific siRNA(A) and LentiCRISPR lentiviral CRISPR/Cas9 (B) methods in HCV FL-Replicon cells alongside appropriate control as indicated. 48h post RTN3 knockdown, cell culture supernatants, and cells were harvested. Total proteins extracted from cells were subjected to western blotting probing for RTN3L&S, NS5A, and NS3 with β-actin serving as an equal loading control. (C) NanoSight NS300 was used to quantify cell released exosomes in cell culture supernatants. (D) Cell released exosomes from LentiCRISPR lentiviral CRISPR/Cas9 knockdown of RTN3L&S conditioning and was co-cultured with naïve Huh7.0 cells (5k Exosomes: 1 Huh7.0 cell) over 72h. Total RNA was then extracted from cells and analyzed by RT-qPCR for HCV RNA using 18s as a housekeeping gene. Data is representative of 3 independently repeat experiments with */#p<0.05 was considered significant by Mann–Whitney U test.
Fig 2: Schematics of RTN3 facilitates the loading of infectious HCV exosomes.(A) Schematic illustration of RTN3 long and short isoforms (RTN3&S) and (B) their cellular endoplasmic reticulum (ER) locations. (C) HCV infection is associated with increased expression of double-stranded (ds) HCV RNA in Huh7 cells. Double-stranded HCV RNA can interact with endoplasmic reticulum located RTN3L&S protein isoforms HCV infected Huh7 cells. Additionally, direct or indirect interactions also occur between other cellular proteins and nucleic acids with dsHCV RNA and RTN3L&S in HCV infectedHuh7 cells. RTN3L&S in HCV infected Huh7 cells can directly modulate the incorporation of replication-competent HCV dsRNA in association with other proteins inside membrane vesicles which are translocated to multivesicular bodies (MVB). This MVB in HCV infected Huh7 cells then fuse with the plasma membrane resulting in the release of infectious viral exosomes. Schematic illustration made use of some smart servier medical art templates (https://smart.servier.com).
Fig 3: Validation the expression pattern of prognostic genes in HCC samples. (A) Western blot analysis of UPB1 (upper panel), SOCS2 (middle panel), and RTN3 (lower panel) in 7 paired normal and tumor tissue samples. (B) Western blot quantitative densitometry of the UPB1, SOCS2, and RTN3 relative expressions. Proteins was quantified and normalized to β-actin. Statistical analysis was done using Wilcoxon rank test. Data are expressed as mean values ± SD. *p < 0.05, **p < 0.01. (C) Representative images from immunohistochemistry (IHC) staining of UPB1 (upper panel), SOCS2 (middle panel), and RTN3 (lower panel) in 82 normal tissues and paired HCC tissues. Left panel: 100 × magnifications; right panel: 200 × magnifications. Bars = 100 μm. (D) Statistical analyses of the average IHC scores of UPB1 (upper panel), SOCS2 (middle panel), and RTN3 (lower panel). Statistical analysis was done using Wilcoxon rank test. Data are expressed as mean values ± SD.
Fig 4: Functional analysis of prognostic genes UPB1, SOCS2, and RTN3 using TCGA dataset. (A) The expression pattern of UPB1 (left panel), SOCS2 (middle panel), and RTN3 (right panel) in 49 HCC tissues and paired adjacent non-tumor tissues from TCGA dataset. The Shapiro-Wilk test was applied to determine whether data followed a normal distribution. The paired t-test was applied to normally distributed data otherwise the Wilcoxon rank test for paired data was applied to assess the expression pattern in HCC tissues. UPB1 and SOCS2 were downregulated in HCC tissues, while RTN3 was upregulated. (B) Association of UPB1 (left panel), SOCS2 (middle panel), and RTN3 (right panel) expression with vascular invasion. Mann-Whitney-Wilcoxon test showed significant differences between vascular invasion group (n = 200) and no vascular invasion group (n = 105) of UPB1 and SOCS2 expression, but not RTN3. (C) Association of UPB1 (left panel), SOCS2 (middle panel), and RTN3 (right panel) expression with the histologic grade. 356 patients with histologic grade information (grade 1, n = 52; grade 2, n = 171; grade 3/4, n = 133) were recruited for the analysis. The Kruskal-Wallis test revealed a negative correlation between UPB1 and SOCS2 expression and histologic grade, while no significant differences was found in RTN3 expression in different groups. (D) Association of UPB1 (left panel), SOCS2 (middle panel), and RTN3 (right panel) expression with pathologic stage. 337 patients with pathologic grade information (stage 1, n = 167; stage 2, n = 82; stage 3/4, n = 88) were recruited for the analysis. The Kruskal-Wallis test was performed. UPB1 and SOCS2 were found to be associated with pathologic stage, while RTN3 was not.
Fig 5: RTN3 overexpression overturns miR-671-5p overexpression-induced effects in IL-1β-induced chondrocytes. A-J Chondrocytes were treated with IL-1β, IL-1β + miR-671-5p or IL-1β + miR-671-5p + RTN3 and their corresponding controls. A Western blot assay was implemented to analyze the protein expression of RTN3 in chondrocytes. B and C Cell proliferation ability was assessed by MTT assay and EdU assay. D and E Flow cytometry was utilized to analyze cell cycle progression and cell apoptosis. F and G The protein levels of PCNA, Cyclin D1, c-caspase 3, MMP3, MMP13 and Collagen II were measured by Western blot assay. H-J Cell inflammatory response was analyzed by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001
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