sj-docx-1-ecx-10.1177_00144029221146574 - Supplemental material for Response Time of Young Children With Complex Communication Needs Following a Communication Opportunity

Supplemental material, sj-docx-1-ecx-10.1177_00144029221146574 for Response Time of Young Children With Complex Communication Needs Following a Communication Opportunity by Tiantian Sun, Ryan Bowles, Sarah N. Douglas and Joshua Plavnick in Exceptional Children</p

DataSheet_1_DNAAF5 promotes hepatocellular carcinoma malignant progression by recruiting USP39 to improve PFKL protein stability.docx

PurposesDynein axonemal assembly factor 5 (DNAAF5) is the transcription factor of regulating the cytoskeleton and hydrodynamic protein complex assembly, however, it was not well elucidated in the malignant progression of hepatocellular carcinoma (HCC).MethodsWe investigated the role of DNAAF5 in hepatocellular carcinoma by using multiple groups of clinical tissues combined with data from the TCGA database. Then we overexpressed DNAAF5 in hepatocellular carcinoma tumor tissues, which correlates with poor patient survival outcomes. Furthermore, we constructed stable cell lines of HCC cells to confirm the cancer-promoting effects of DNAAF5 in hepatocellular carcinoma. To explore the mechanisms of DNAAF5, transcriptome sequencing combined with mass spectrometry was also performed, which showed that DNAAF5 affects its downstream signaling pathway by interacting with PFKL and that DNAAF5 regulates PFKL protein stability by recruiting the deubiquitination protein, USP39. To corroborate these findings, the same series of tissue microarrays were used to confirm correlations between DNAAF5 and PFKL expressions. In animal experiments, DNAAF5 also promoted the proliferation of HCC cells.ResultsWe found that DNAAF5 expressions were markedly higher in HCC tissues, compared to the adjacent normal tissues. Increased levels of DNAAF5 were associated with significantly worse prognostic outcomes for HCC patients. Cell function experiments showed that HCC cells of overexpressing DNAAF5 exhibited faster proliferation rates, stronger clone formation abilities and higher drug resistance rates. However, tumor cell proliferation rates and colony formation were significantly decreased after DNAAF5 knockout, accompanied by an increase in sensitivity to sorafenib. In addition, the results of our study showed that DNAAF5 accelerates PFKL protein deubiquitination by recruiting USP39 in HCC cells. Furthermore, The overexpression of DNAAF5 could promote HCC cell proliferation in vivo and in vitro, whereas USP39 knockdown inhibited this effect. Overall, DNAAF5 serves as a scaffold protein to recruit USP39 to form a ternary complex by directly binding the PFKL protein, thereby improving the stability of the latter, which promotes the malignant process of hepatocellular carcinoma.ConclusionsThese findings revealed DNAAF5 was negatively correlated with the prognosis of patients with hepatocellular carcinoma. It underlying mechanism showed that DNAAF5 directly binds PFKL and recruits the deubiquitinated protein (USP39) to improve the stability of the PFKL protein, thus enhancing abnormal glycolysis in HCC cells.</p

List of alignment results of proteins related to TJ and AJ.

<p>T stands for the sequence eligible the conditions and F stands for the opposite.</p>*<p>, indicated this gene had been predicted by BLASTx searches in Swissprot/NR protein database. OV<i>: O. vulgaris</i>, AC<i>: A. californica</i>, SG<i>: S. gregaria</i>, HM<i>: H. medicinalis</i>.</p

Phylogenetic tree of synaptotagmin-7.

<p>Phylogenetic tree of synaptotagmin-7.</p

Primers for 14 target genes and β-actin gene used for Real-time PCR.

<p>Primers for 14 target genes and β-actin gene used for Real-time PCR.</p

List of top 20 sequences sorted by reads.

<p>List of top 20 sequences sorted by reads.</p

GO categories with highest number of sequences corresponding to CNS function.

<p>GO categories with highest number of sequences corresponding to CNS function.</p

Distribution of second level GO annotation in three categories.

<p>Distribution of second level GO annotation in three categories.</p

Comparison between <i>O. vulgaris</i> dataset and four different species CNS datasets using BLASTn and tBLASTx.

<p>Comparison between <i>O. vulgaris</i> dataset and four different species CNS datasets using BLASTn and tBLASTx.</p

Phylogenetic tree of synaptophysin.

<p>Phylogenetic tree of synaptophysin.</p