Journal: Cell Death & Disease

Article Title: Hypoxia-induced shift in the phenotype of proteasome from 26S toward immunoproteasome triggers loss of immunoprivilege of mesenchymal stem cells

doi: 10.1038/s41419-020-2634-6

Figure Lengend Snippet: To determine the immunogenicity of MSCs, normoxic and hypoxic human MSCs (with or without immunoproteasome inhibitor- Onx0914 1 µM for 4 h) were co-cultured with allogeneic leukocytes at a ratio 1:10 for 72 h. a Leukocyte mediated cytotoxicity in MSCs (LDH release) increased significantly in hypoxic MSCs vs. normoxic cells, which was rescued by inhibition of immunoproteasome. n = 5. b The effect of MSCs on leukocyte proliferation was measured using WST1 proliferation assay kit. After 72 h of co-culture, normoxic MSCs were able to decrease leukocyte proliferation compared to control (PHA treated leukocytes). However, hypoxia treated MSCs had no effect on leukocyte proliferation, immunoproteasome inhibited hypoxic MSCs significantly decreased leukocytes proliferation. n = 10. c After 72 h of co-culture, the effect of MSCs on CD4 + CD25 + FOXP3 + Treg cell induction in a mixed leukocyte population was assessed by flow cytometry. The number of Treg cells decreased after co-culture with hypoxic MSCs. However, co-culture with immunoproteasome inhibited hypoxic MSCs increased the number of Treg cells. n = 3. * p < 0.05 compared to normoxic MSC; @ p < 0.05 compared to PHA group; # p < 0.05 compared to hypoxic MSCs, each experiment was repeated 3–4 times.

Article Snippet: After 72 h of co-culture, leukocyte-mediated cytotoxicity in MSCs was determined by measuring the lactate dehydrogenase (LDH) which was released from the damaged MSCs (LDH Cytotoxicity Detection Kit; Clontech).

Techniques: Cell Culture, Inhibition, Proliferation Assay, Co-Culture Assay, Flow Cytometry

Journal: Journal of Clinical Medicine

Article Title: Gene Expression Changes Associated with Nintedanib Treatment in Idiopathic Pulmonary Fibrosis Fibroblasts: A Next-Generation Sequencing and Bioinformatics Study

doi: 10.3390/jcm8030308

Figure Lengend Snippet: ( A ) Morphological changes of idiopathic pulmonary fibrosis (IPF) fibroblasts after nintedanib treatment. The human IPF lung fibroblasts were treated with 0.1% DMSO (control), 1 µM, 2 µM, and 4 µM nintedanib. The cell morphological changes were observed with an inverted-phase contrast microscope at 24 h, 48 h, and 72 h after nintedanib treatment. ( B ) Inhibition effects of nintedanib on cell proliferation of IPF fibroblasts. The human IPF lung fibroblasts were treated with 0.1% DMSO (control), 1 µM, 2 µM, and 4 µM nintedanib. Cell proliferation was measured with the WST-1 cell proliferation assay at 24 h, 48 h, and 72 h after nintedanib treatment. * p < 0.05; ** p < 0.01; *** p < 0.001. ( C ) Effects of nintedanib on apoptosis of IPF fibroblasts. The human IPF lung fibroblasts were treated with 0.1% DMSO (control), 1 µM, 2 µM, and 4 µM nintedanib. Cell apoptosis was measured with the Annexin V/Propidium Iodide flow cytometry at 24 h, 48 h, and 72 h after nintedanib treatment.

Article Snippet: Cell apoptosis was assessed using the Annexin V-FITC Early Apoptosis Detection Kit (Cell signaling Technology, Beverly, MA, USA.

Techniques: Control, Microscopy, Inhibition, Proliferation Assay, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 is overexpressed in OSCCs patient samples and OSCC-derived cell lines. Total RNA from fresh samples and cell lines was converted to cDNA and subjected to qPCR. For gene expression analysis of tissue samples relative quantification was based on the comparison of a pool of five normal oral tissues, while the spontaneously immortalized but non-transformed epithelial cell line HGK was used as reference for comparison with OSCC-derived cell lines. The levels of HPSE1 mRNA were significantly higher in OSCC cell lines compared with HGK cells (A) . The high expression levels of protein HPSE1 were confirmed on OSCC-derived cell lines by Western Blot analysis (B) . The levels of HPSE1 mRNA were also significantly higher in OSCC tissue samples compared to normal oral mucosa (C) . Representative images in a high-power field (200×) of immunohistochemical analysis for HPSE1 in Normal oral tissue (D) and OSCC tissue preparations confirmed its higher expression at protein level; with a distinct cytoplasmic distribution and intensity in oral cancer samples expressing both higher (E) and lower (F) levels of HPSE1. Results were statistically determined by ANOVA followed by Tukey multiple comparison test, where ** p < 0.005, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Derivative Assay, Expressing, Transformation Assay, Western Blot, Immunohistochemical staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 overexpression discriminates OSCC and normal samples and predicts low patient survival. (A) Kaplan–Meier cumulative curves for disease-free survival of patients with OSCC as a function of HPSE1 expression, showing a 70% probability of death in patients with higher expression of HPSE1 compared with those with low expression. (B) Receiver operating characteristic (ROC) curves showing the ability of the HPSE1 overexpression distinguishes OSCC tumors from normal oral samples. (C) Kaplan–Meier of multivariate survival analysis combining HPSE1 expression levels with pT stages, where score 1: HPSE1 lower expressing samples associated with pT1/2, score 2: HPSE1 lower expressing samples associated with pT3/4 or higher HPSE1 with pT1/2, and score 3: HPSE1 higher expressing samples associated with pT3/4. These results confirmed that HPSE1 overexpression was significantly associated with higher grade tumors.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 knockdown and upregulation efficiency in OSCC cells. The efficiency of endogenous HPSE1 modulation was verified by Immunofluorescence (A) , Western Blot (B) and RT-qPCR (C) analyzes. For the loss-of-function strategy, SCC-9 cells were transduced with shRNA expressing a vector sequence against HPSE1 (shRNA HPSE1−) and empty vector (scrRNA control). For the gain-of-function strategy, SCC-9 cells were transduced with a ORF clone in a shuttle vector to enhance HPSE1 expression (orfRNA HPSE1+), along with mock-transduced cells as described in Methods . shRNA HPSE1− cells showed a significant reduction and orfRNA HPSE+ cells showed a significant upregulation of HPSE1 in both mRNA and protein levels compared to scrRNA control and parental mock-transduced SCC-9 control cells (pSCC9), without targeting sequences. Results were statistically determined by ANOVA, followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Immunofluorescence, Western Blot, Quantitative RT-PCR, Transduction, shRNA, Expressing, Plasmid Preparation, Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Downregulation of HPSE1 inhibits proliferation and enhances apoptosis of OSCC cells. Cells were subjected to MTT cell proliferation (A) , DNA content cell cycle analysis (B) , and apoptosis (C) assays. (A) Cell Proliferation Assay of the OSCC SCC9 cell line (Control) compared to empty vector SCC9 control clone (ScrRNA control), HPSE1 inhibitory/silencing SCC9-ShRNA HPSE1− clone, and the HPSE1 overexpressing SCC9-OrfRNA HPSE1+ clone. (B) Quantification of cell cycle analysis was performed by flow cytometry after staining with propidium iodide for the SCC-9 control cells and the respectives HPSE1-modulated clones. Abrogation of HPSE1 induced arrest of the SCC-9 cells cycle in G1 phase, and its upregulation significantly induced cell proliferation. (C) Flow cytometric analysis of apoptosis showed a remarkable increase in the number of apoptotic cells in HPSE1-silenced cells (ShRNA HPSE1−), while SCC-9 overexpressing HPSE1 (Orf-RNA HPSE1+) exhibited a reduction in apoptosis. (D) No significant differences in cell adhesion properties were observed among any transduced clones modulating HPSE1 expression, compared to parental mock-transduced cells (control). Plots compose experimental triplicate analysis and were statistically calculated using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: MTT Cell Proliferation, Cell Cycle Assay, Proliferation Assay, Plasmid Preparation, shRNA, Flow Cytometry, Staining, Clone Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Overexpression of HPSE1 is associated with migration, invasion, ECM remodeling, and acquisition of EMT properties. (A) Photomicrographs of cell lines were taken 0, 24, and 48 h after wounding (40X). The average width of the lacunae was measured. Migration of SCC-9 cells was significantly decreased in HPSE1-silenced cells (ShRNA HPSE1−) and increased in HPSE1-upregulated clones (OrfRNA HPSE1+). Migration analysis based on this assay showed that cells with lower expression of HPSE1 closed the scratch wound significantly slower than the SCC9 Control cells. (B) Invasion of SCC-9 cells was significantly inhibited by HPSE1 knockdown, and significantly enhanced after its upregulation. (C) Analysing EMT markers, the downregulation of HPSE1 significantly induced the expression of E-Cadherin (E-CAD), while OrfRNA HPSE1+ SCC9 clones overexpressing heparanase had a significant increasing of Vimentin (VIM) and SNAIL expressions. (D) The upregulation of HPSE1 significantly enhanced the expressions of MMP2 and MMP9. All the graphs compile experimental triplicate analyses, and the results were obtained by ANOVA followed by Tukey assay, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Migration, shRNA, Clone Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Overexpression of HPSE1 enhances tumor neo-vascularization and induces VEGFA expression. HUVEC cells on a Miogel 2D layer co-culture. Photomicrograph of HUVEC endothelial cells added with preconditioned medium from OSCC cell lines and their respective clones after 12 h of experiment: (A) HUVEC cells incubated with preconditioned medium of SCC9-OrfRNA HPSE1+ clone overexpressing HPSE1 (100×); (B) HUVEC cells incubated with preconditioned medium of parental SCC9 (Control) (100×); (C) HUVEC cells incubated with preconditioned medium of SCC9-ShRNA HPSE1- clone with reduction of HPSE1 expression (100×). (D) Quantification of measures of the vessel circumference perimeter for each one of the conditions; the graph compiles two experimental triplicate analysis. (E) Gene expression analysis of endothelial growth factor VEGFA by qRT-PCR; the graph compiles folded expression values of relative quantification by ddCT obtained through comparison of clones OrfRNA HPSE1+ and ShRNA HPSE1− relative to the control SCC9 cells (normal reference = 1). Normalization of the analysis was performed using the endogenous control gene, PPIA. Statistical tests were performed using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Expressing, Co-Culture Assay, Clone Assay, Incubation, shRNA, Quantitative RT-PCR