Fig 1: PDCD2 forms a complex with the free uS5 protein in human cells. (A) Scatterplot showing results from GFP-PDCD2 affinity purification-coupled to mass spectrometry (AP-MS) plotted by SILAC ratio on the x-axis (peptide intensity originating from the GFP-PDCD2 purification versus the GFP control), which reflects specificity; and relative peptide intensity up the y-axis (total peptide intensity for each protein), reflecting the relative abundance of each protein in the purification. (B) Scatterplot showing results from proximity-dependent biotinylation (PDB) assay using BirA-PDCD2 coupled to SILAC-based mass spectrometry (MS) quantifications, as described in (A). (C) Venn diagram showing overlap between PDCD2-associated proteins identified by AP-MS (n = 64) and PDB-MS (n = 8). (A–C) The four proteins common to AP-MS and PDB-MS are shown in color. (D) Western blot analysis of cell extracts (lanes 1–2) as well as uS5 (lanes 5–6) and IgG (lanes 3–4) immunoprecipitates prepared using extracts from HeLa cells that conditionally express control (lanes 1, 3 and 5) and PDCD2-specific (lanes 2, 4 and 6) shRNAs. Antibodies used for Western blotting are indicated on the right. The asterisk (top panel) indicates the presence of a non-specific protein detected using the PDCD2 antibody. (E) Western blot analysis of total extracts (Input, lanes 1–2) and anti-Flag precipitates (IP, lanes 3–4) prepared from E. coli that co-expressed PDCD2 and uS5 (lanes 2 and 4) or that expressed PDCD2 alone (lanes 1 and 3). (F) Western analysis of the indicated proteins (right) using fractions of centrifuged sucrose gradients that were prepared using extracts of HeLa cells. The positions of the 40S, 60S/80S, and polysomes sedimentation are indicated at the bottom.
Fig 2: PDCD2 increases sorafenib-induced apoptosis and decreases cell metastatic rate in the HepG2/SF cell line. (A) Overexpression of PDCD2 enhanced sorafenib induced apoptosis, determined by flow cytometry. (B) PDCD2 decreased the metastatic rate of the HepG2/SF cell line (magnification, ×20). (C) Western blotting demonstrates increased expression of cleaved caspase 3 and reduced expression of MMP2 and MMP9 following transfection with PDCD2. The data are presented as the mean ± standard deviation of three independent tests. *P<0.05, **P<0.01. HepG2/SF, sorafenib-resistant HepG2 cells; PDCD2, programmed cell death domain 2; MMP, matrix metalloproteinase; PI, propidium iodide; FITC, fluorescein isothiocyanate.
Fig 3: PDCD2 significantly decreases the MDR phenotype in the HepG2/SF cell line. (A) Overexpression of PDCD2 significantly reduced the IC50 value for sorafenib in the HepG2/SF cell line. (B) Western blot demonstrates transfection with PDCD2 decreased the expression of MDR1 in the HepG2/SF cell line. (C) Quantification of protein expression. Data are presented as the mean ± standard deviation of three independent tests. *P<0.05, ***P<0.001. HepG2/SF, sorafenib-resistant HepG2 cells; PDCD2, programmed cell death domain 2; MDR, multidrug resistance; IC50, half maximal inhibitory concentration.
Fig 4: PDCD2 binds to uS5 in a cotranslational manner. (A) Western blot analysis of total extracts (lanes 1–3) and anti-GFP precipitates (lanes 4–6) prepared from HeLa cells that stably express GFP (lanes 1 and 4), GFP-PDCD2 (lanes 2 and 5), and GFP-PDCD2L (lanes 3 and 6). (B) Fold mRNA enrichment (IP/input ratio) for the indicated mRNAs in GFP, GFP-PDCD2, and GFP-PDCD2L immunoprecipitates was analyzed by RT-qPCR and normalized to a control housekeeping mRNA (GAPDH). Values were then set to 1.0 for the control GFP purification. *P = 0.05; Student's t-test. (C) Western blot analysis of total extracts (lanes 1–3) and anti-GFP precipitates (lanes 4–6) prepared from HeLa cells that stably express GFP (lanes 1 and 4), GFP-PDCD2 wild-type (lanes 2 and 5), and GFP-PDCD2 TYPP mutant (lanes 3 and 6). (D) Analysis of mRNA enrichment as described in panel B, but using GFP, GFP-PDCD2 WT, and GFP-PDCD2 TYPP immunoprecipitates. *P = 0.05, Student's t-test. (E) Western blot analysis of total extracts prepared from U2OS cells that were previously transfected with the indicated DNA constructs for 24 h. The blots were analyzed using antibodies specific for PDCD2 (top), uS5 (middle), and Tubulin (bottom). (F) Representative BiFC images showing interaction between VN-PDCD2 and uS5-VC in living human cells. U2OS cells that co-expressed uS5-VC with either the wild-type VN-PDCD2 (a–d) or the VN-PDCD2 TYPP mutant (e-h) were fixed and simultaneously analysed by direct fluorescence (b and f) and immunostaining for the nucleolar marker fibrillarin (c and g). DNA staining with DAPI shows the nucleus of each cell (a and e).
Fig 5: PDCD2 inhibits EMT and MDR phenotypes through suppression of Snail. (A) Western blotting and (B) quantification of the results demonstrated that PDCD2 decreased the expression of Vimentin and Snail and increased the expression of E-cadherin, indicating that PDCD2 may regulate EMT by the suppression of Snail. (C) Western blotting and (D) quantification of the results suggested that Snail-sh significantly decreased the expression of Snail. (E) Co-transfection with PDCD2 and Snail-sh plasmids in HepG2/SF cells blunted the function of PDCD2, indicating that PDCD2 reversed MDR in a Snail-dependent manner. (F) Co-transfection with PDCD2 and Snail-sh plasmids in HepG2/SF cells partially eliminated the EMT phenotype. Data are presented as the mean ± standard deviation of three independent tests. *P<0.05, ***P<0.001. HepG2/SF, sorafenib-resistant HepG2 cells; PDCD2, programmed cell death domain 2; MDR, multidrug resistance; EMT, epithelial-mesenchymal transition; IC50, half maximal inhibitory concentration; sh, small hairpin.
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