Journal: bioRxiv

Article Title: HCP5 prevents ubiquitination-mediated UTP3 degradation to inhibit apoptosis by activating c-Myc transcriptional activity

doi: 10.1101/2022.06.13.495862

Figure Lengend Snippet: A Volcano plots showing upregulated and downregulated ncRNAs in KYSE450/DDP and YES2/DDP cells compared to their parental cells. B RT–qPCR detection of HCP5 expression in the parental and DDP-resistant cell models. The data are presented as the means ± s.d.; two-tailed t test, *** P < 0.001; n = 3. C RT–qPCR detection of HCP5 expression in KYSE30 and KYSE450 cells treated with DDP for 0, 6, 12, and 24 h. The data are presented as the means ± s.d.; two-tailed t test, *** P < 0.001, ** P < 0.01, * P < 0.05; n = 3. D Overall survival of ESCC patients with low and high HCP5 expression levels (divided by the mean expression value). Kaplan–Meier survival plots are shown. E Statistical analysis of HCP5 expression in 168 paired ESCC tissues and adjacent normal tissues. Box plot representation: from top to bottom—maximum, 75th percentile; median, 25th percentile; and minimum values; two-tailed t test. F Statistical analysis of HCP5 expression in 53 paired ESCC tissues and adjacent normal tissues in the GEO database. Box plot representation: from top to bottom—maximum, 75th percentile; median, 25th percentile; and minimum values; two-tailed t test. G-J Tumor volumes and representative images (G and I) and tumor weights (H and J) of the xenografts derived from the indicated cells. The data are presented as the means ± s.e.m.; two-tailed t test, *** P < 0.001, ** P < 0.01; n = 7. K-M Tumor weights in the three PDX models from ESCC patients treated with AAV-shCtrl or AAV-shHCP5. The data are presented as the means ± s.e.m.; two-tailed t test, *** P < 0.001; n = 8.

Article Snippet: For ISH, human ESCC tissue microarray slides were purchased from Servicebio (#G6040, Wuhan, Hubei, China).

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: HCP5 prevents ubiquitination-mediated UTP3 degradation to inhibit apoptosis by activating c-Myc transcriptional activity

doi: 10.1101/2022.06.13.495862

Figure Lengend Snippet: A FISH detection of HCP5 localization in KYSE150 and KYSE510 cells. Nuclei were stained with Hoechst 33342. Scale bar, 30 μm. B List of HCP5-interacting proteins identified using MS with KYSE150 and KYSE510 cells. C Western blot analyses of UTP3 in precipitates from RNA pull-down assays performed using antisense or sense sequences of HCP5. D RT–qPCR measurement of the HCP5 level in precipitates from RIP assays performed using IgG or anti-UTP3 antibodies. The data are presented as the means ± s.d.; two-tailed t test, *** P < 0.001; n = 3. E Correlation analysis of HCP5 and UTP3 protein expression in ESCC cell lines. Spearman correlation coefficients are shown. F Western blot analyses of UTP3 and MCL1 expression in control and HCP5-silenced KYSE150 and KYSE510 cells treated with DMSO or MG132 for 8 h. G Western blot analyses of UTP3 expression in control and HCP5-silenced KYSE150 and KYSE510 cells subjected to a CHX pulse-chase assay. H Quantitative analyses of UTP3 expression in control and HCP5-silenced KYSE150 and KYSE510 cells subjected to the CHX pulse-chase assay. Quantification of UTP3 expression relative to β -actin expression is shown. The data are reported as the means ± s.e.m.; n = 3. I, J Western blot detection to assess UTP3 ubiquitination in control and HCP5-silenced KYSE150 (I) and KYSE510 cells (J).

Article Snippet: For ISH, human ESCC tissue microarray slides were purchased from Servicebio (#G6040, Wuhan, Hubei, China).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Two Tailed Test, Expressing, Pulse Chase

Journal: bioRxiv

Article Title: HCP5 prevents ubiquitination-mediated UTP3 degradation to inhibit apoptosis by activating c-Myc transcriptional activity

doi: 10.1101/2022.06.13.495862

Figure Lengend Snippet: A, B GSEA results based on RNA-seq data comparing HCP5-silenced to control KYSE150 (A) and KYSE510 (B) cells. C Prediction of the sequence logo of the MYC-binding site based on the JASPAR database. D Predicted binding sequence of MYC in the VAMP3 promoter region, as determined from the JASPAR database. E Schematic showing MYC binding sites at the VAMP3 promoter region and the unique peaks identified by ATAC-seq. F, G ChIP analyses of the indicated cells using antibodies against c-Myc to identify the indicated gene promoter segments. The data are presented as the means ± s.d.; two-tailed t test, *** P < 0.001, ** P < 0.01; n = 3. H RT–qPCR detection of the expression of the indicated genes in samples from ESCC patients with chemoresistance or chemosensitivity. The data are presented as the means ± s.e.m.; two-tailed t test; n = 17 for chemoresistant patients and n = 17 for chemosensitive patients. I Correlation analyses between the indicated gene expressions in ESCC samples. Spearman correlation coefficients are shown.

Article Snippet: For ISH, human ESCC tissue microarray slides were purchased from Servicebio (#G6040, Wuhan, Hubei, China).

Techniques: RNA Sequencing Assay, Sequencing, Binding Assay, Two Tailed Test, Quantitative RT-PCR, Expressing