DOI: 10.5061/dryad.8cz8w9gzp
title: Panaxatriol exerts anti-senescence effects and alleviates osteoarthritis and cartilage repair fibrosis by targeting UFL1

description: Osteoarthritis is the most commmon degenerative joint disease which can
 lead to disability eventually. However, there are currently no safe and
 effective interventions available. Therefore, it is urgent to develop
 effective drugs that can reduce cartilage damage and treat OA.With the in
 vitro culturing of human cartilage explants and mouse OA model, it was
 found that, panaxtriol, a natural small molecule drug, could promote
 chondrocyte anabolism and inhibit catabolism. It could also reduce the
 loss of cartilage matrix , decrease the subchondral osteosclerosis, and
 relieve pain in mice. Eventually, it could delay the progression of OA.
 Subsequently, the binding target of panaxtriol was found to be UFL1
 through the target stability of drug affinity reaction (DARTS). The UFL1
 knockout cell line was constructed using CRISPR-Cas9. Then, the regulation
 of chondrocyte metabolism by panaxtriol was found to dependent on UFL1.
 Transcriptome sequencing of UFL1 knockout cells were conducted. Through
 differential gene analysis, GO analysis and KEGG analysis, it showed that
 UFL1 was closely related to cell senescence. By using activators and
 inhibitors of the signaling pathway, panaxtriol was proved that it can
 inhibit chondrocyte senescence through UFL1/FOXO1/P21 and UFL1/NF-κB/SASPs
 signaling pathways to delay the progression of OA. It also could inhibit
 the formation of fibrocartilage during cartilage repair by UFL1/FOXO1/COL1
 signaling pathway. Lastly, a sustained release system of panaxtriol was
 constructed based on PLGA-PEG. Through drug sustained release, the
 therapeutic effect was achieved while the number of intra-articular
 injection was reduced, thereby alleviating joint swelling and joint
 injury.
description: <strong>RNA sequencing: </strong>C28/I2 cells and
 UFL1 knock-out cells were send to RNA sequencing analysis, each group has
 3 repeats. According to the technology vendor's process, RNA
 integrity was assessed using the RNA Nano 6000 Assay Kit of the
 Bioanalyzer 2100 system (Agilent Technologies, CA, USA). After the
 construction of the library, the library was initially quantified by
 Qubit2.0 Fluorometer, then diluted to 1.5ng/ul, and the insert size of the
 library is detected by Agilent 2100 bioanalyzer. After the library is
 qualified, the different libraries are pooling according to the effective
 concentration and the target amount of data off the machine, then being
 sequenced by the Illumina NovaSeq 6000. Raw data (raw reads) of fastq
 format were firstly processed through fastp software to get clean data
 with high quality. We selected Hisat2 as the mapping tool to read mapping
 to the reference genome. FeatureCounts (v1.5.0-p3) was used to count the
 reads numbers mapped to each gene. And then FPKM of each gene was
 calculated based on the length of the gene and reads count mapped to this
 gene. Differential expression analysis of two groups was performed using
 theDESeq2 R package (1.20.0). padj&lt;=0.05 and |log2(foldchange)|
 &gt;= 1 were set as the threshold for significantly differential
 expression. Gene Ontology (GO) enrichment analysis of differentially
 expressed genes wasimplemented by the clusterProfiler R package (3.8.1),
 in which gene length biaswascorrected. GO terms with corrected Pvalue less
 than 0.05 were consideredsignificantly enriched by differential expressed
 genes. We used clusterProfiler R package (3.8.1) to test the statistical
 enrichment of differential expression genes in KEGG pathways. Reactome
 pathways with corrected Pvalue less than 0.05 were considered
 significantly enriched by differential expressed genes. We use the local
 version of the GSEA analysis tool
 http://www.broadinstitute.org/gsea/index.jsp.
description: **Panaxatriol exerts anti-senescence effects and alleviates osteoarthritis
 and cartilage repair fibrosis by targeting UFL1**
 [https://doi.org/10.5061/dryad.8cz8w9gzp](https://doi.org/10.5061/dryad.8cz8w9gzp) **Quantification of gene expression level** feature Counts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels. **Differential expression analysis** Differential expression analysis of two conditions/groups (three biological replicates per condition) was performed using the DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value &lt;=0.05 found by DESeq2 were assigned as differentially expressed. The significant differences in each gene in all comparison combinations are shown in this dataset. **Column A：gene_id:** gene ID **Column B：NC_1_count:** The original readcount value of NC1 sample which is normal C28/I2 cells. **Column C：NC_2_count:** The original readcount value of NC2 sample which is normal C28/I2 cells. **Column D：NC_3_count:** The original readcount value of NC3 sample which is normal C28/I2 cells. **Column E：UFL1_KO_1_count:** The original readcount value of UFL1-KO1 sample which is UFL1 konck-out C28/I2 cells. **Column F：UFL1_KO_2_count:** The original readcount value of UFL1-KO2 sample which is UFL1 konck-out C28/I2 cells. **Column G：UFL1_KO_3_count:** The original readcount value of UFL1-KO3 sample which is UFL1 konck-out C28/I2 cells. **Column H：NC_1 \_fpkm:** The fpkm value of NC1 sample which is normal C28/I2 cells. **Column I：NC_2 \_fpkm:** The fpkm value of NC2 sample which is normal C28/I2 cells. **Column J：NC_3 \_fpkm:** The fpkm value of NC3 sample which is normal C28/I2 cells. **Column K：UFL1_KO_1 \_fpkm:** The fpkm value of UFL1-KO1 sample which is UFL1 konck-out C28/I2 cells. **Column L：UFL1_KO_1 \_fpkm:** The fpkm value of UFL1-KO1 sample which is UFL1 konck-out C28/I2 cells. **Column M：UFL1_KO_1 \_fpkm:** The fpkm value of UFL1-KO1 sample which is UFL1 konck-out C28/I2 cells. **Column N：UFL1_KOvsNC_UFL1_KO:** the average of standardized readcounts for UFL1** **konck-out group **Column O：UFL1_KOvsNC_NC:** the mean of the standardized readcount of the control (NC) group. **Column P：UFL1_KOvsNC_log2FoldChange:** the ratio of gene expression levels between UFL1 knock-out group and NC group, with a base of 2 for the logarithm, the symbol "-" in this column means minus. **Column Q：UFL1_KOvsNC_pvalue:** the p-value of a significant test for the comparison between UFL1 knock-out group and NC group. **Column R：UFL1_KOvsNC_padj:** the corrected p-value of a multiple hypothesis test for the comparison between UFL1 knock-out group and NC group. **Column S：gene_name:** gene name, the symbol "-" in this column means none. **Column T：gene_chr:** the name of the chromosome where the gene is located **Column U：gene_start:** The starting position of the gene on the chromosome **Column V：gene_end:** The end position of the gene on the chromosome **Column W：gene_strand:** information about the positive(+) and negative(-) strands of the chromosome where the gene is located **Column X：gene_length:** gene length, the sum of all non-overlapping regions from the start to the end of the gene **Column Y：gene_biotype:** gene type, such as protein-coding genes, long non-coding genes, etc. **Column Z：gene_description:** gene function description, the first && symbol represents the reference genome annotation information, the first && symbol represents the swissprot database annotation, and the second && symbol represents the pfam database annotation **Column AA：gene_tf_family:** gene transcription factor family annotation