Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet: Humanin-induced chemoresistance requires ATR signaling (A) hGBM1 cells were stimulated with HN or vehicle (Ctrl.), underwent transcriptomics, and differentially expressed genes (DEGs) were analyzed by bioinformatics. (B) Experiments described in (A) were repeated with hGBM-1, 2, and 3 cells providing 12 consistent DEGs, of which several components assembled in a network. (C) HUS1 was associated with outcome in human GBMs. (D) In a myeloid-free brain sample, hGBMs have a basal level of HUS1 expression, which is upregulated by interaction with hiPSC microglia in a GP130-dependent manner. (E and F) Contribution of the ATR pathway to humanin-induced GBM expansion (E) and chemoresistance (F) was demonstrated with the ATR inhibitor AZ20. (G) Western blots showing expression levels of HUS1, ATR and beta-actin (loading control) and a readout for of ATR activation (pT1989) in hGBM1 cells treated with bovine serum albumin (control), TMZ, HN, or AZ20. (H) In summary, AZ20 does not cooperate with TMZ per se, but blocks HN-induced TMZ resistance. The number of biological replicates is indicated (dots in graphs indicate data from individual experiments); all error bars are presented as mean ± SDM. Statistical significance is shown as FDR in (A), one-way ANOVA (D, E), or two-way ANOVA (F): ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; NS, not significant.

Article Snippet: HUS1 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-3364-pZIP-hCMV-ZsGreen-GVO-TRI.

Techniques: Expressing, Western Blot, Control, Activation Assay

Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet: Humanin-induced chemoresistance can be blocked therapeutically (A) Tumor size of orthotopic HN-WT or HN-C8a tumors was compared in mice receiving TMZ or vehicle (in animals with established tumor growth, 5x per week for 2 weeks; pre-defined endpoint was at 3 weeks). (B) Orthotopic hGBM1 was infused with HN (100 nM) or vehicle (artificial cerebrospinal fluid, aCSF) and i.p. injected with bazedoxifene-A (5 injections of BZA per week; 40 mg/kg; for 2 weeks) or vehicle; brains were labeled for HUS1; HUS1 expression was quantified. (C) Mice with established, orthotopic HN-WT tumors received TMZ (50 mg/kg) and were cotreated with vehicle or BZA (as in B); after 3 weeks, tumor size was quantified (dashed line: average data from untreated WT GBMs). (D) Mice with HN-WT GBMs received TMZ and were cotreated with vehicle or BZA (as in C); GBM samples were immunostained for active caspase-3 and immunolabeling was quantified (dashed line: average data from untreated WT GBMs). (E) Intratumoral vascularization and vessel diameter were compared in HN-WT or HN-C8a tumors receiving TMZ. (F) The HN-WT GBM mouse model was i.p. injected with TMZ and cotreated either with BZA or vehicle and the extent of intratumoral vascularization was compared. The number of biological replicates is indicated (dots in graphs indicate data from individual mice); all error bars are presented as mean ± SDM. Statistical significance is shown by one-way ANOVA (A, E), two-way ANOVA (B–D), or t test (F): ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; NS, not significant. Scale bars in (B, C) indicate 1 mm; scales in (D) represent 500 (overview) or 10 μm (magnified).

Article Snippet: HUS1 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-3364-pZIP-hCMV-ZsGreen-GVO-TRI.

Techniques: Injection, Labeling, Expressing, Immunolabeling

Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet:

Article Snippet: HUS1 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-3364-pZIP-hCMV-ZsGreen-GVO-TRI.

Techniques: Plasmid Preparation, Recombinant, Transfection, Fluorescence, Staining, Reverse Transcription, Expressing, Liposomes, Mutagenesis, shRNA, Control, Construct, Software, Imaging, Functional Assay, Dissection, Sequencing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Cell Physiology

Article Title: Abundance of TRPC6 protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes

doi: 10.1152/ajpcell.00014.2011

Figure Lengend Snippet: Changes in the planar surface area of MCs in response to ANG II. A and B: representative photomicrographs of MCs used in contraction assays. A: MCs were cultured in NG (5.6 mM d-glucose + 20 mM l-glucose) and were transfected with either enhanced green fluorescent protein (EGFP) alone or EGFP plus shRNA construct specific for human TRPC6 (EGFP + hTRPC6-shRNA). B: MCs were cultured in HG (25.6 mM d-glucose) and were transfected with either EGFP alone or EGFP tagged TRPC6 expression plasmid (TRPC6-EGFP). The green cells indicate the positively transfected MCs and were used for analysis indicated by arrows. C: summary data from the experiments presented in A (Ca) and B (Cb) showing the contractile response of MCs with different treatments, calculated as [(the surface area of MCs after ANG II − the surface area of MCs before ANG II)/the surface area of MCs before ANG II] × 100%. n indicates the number of cells analyzed in each group. *P < 0.05; **P < 0.01 compared with EGFP group.

Article Snippet: As described by Du et al. ( 7 ) with modifications, human MCs were plated on a 22 × 22 mm coverslip and were transiently transfected with either green fluorescent protein (GFP) + hTRPC6-short hairpin RNA (shRNA) constructs (1:10, GFP:hTRPC6-shRNA) or TRPC6-enhanced GFP (EGFP) constructs using GenJet (SignaGen, Gaithersburg, MD).

Techniques: Cell Culture, Transfection, shRNA, Construct, Expressing, Plasmid Preparation

Journal: Molecular Cancer

Article Title: LncRNA PTAR promotes EMT and invasion-metastasis in serous ovarian cancer by competitively binding miR-101-3p to regulate ZEB1 expression

doi: 10.1186/s12943-018-0870-5

Figure Lengend Snippet: The identification of a ceRNA network for mesenchymal OvCa. a The ceRNA network in mesenchymal OvCa. Rounded rectangles, ellipses and hexagons denote lncRNAs, EMT genes and miRNAs, respectively. Up-regulated lncRNAs, EMT genes and miRNAs are shown in red and down-regulated lncRNAs, EMT genes and miRNAs are shown in green. b ZEB1 was significantly up-regulated in mesenchymal OvCa compared with epithelial OvCa in the TCGA data set. c Positive correlation between PTAR and ZEB1 expression in the TCGA data set. d The lncRNA PTAR was significantly up-regulated in mesenchymal OvCa compared with epithelial OvCa in the TCGA data set. e Nagative correlation between miR-101 and ZEB1 expression in the TCGA data set. f Nagetive correlation between PTAR and miR-101 expression in the TCGA data set. g miR-101 was significantly down-regulated in mesenchymal OvCa compared with epithelial OvCa in the TCGA data set

Article Snippet: The lentiviral constructs of PTAR shRNA were constructed by Biowit Technology (Shenzhen, China).

Techniques: Expressing

Journal: Molecular Cancer

Article Title: LncRNA PTAR promotes EMT and invasion-metastasis in serous ovarian cancer by competitively binding miR-101-3p to regulate ZEB1 expression

doi: 10.1186/s12943-018-0870-5

Figure Lengend Snippet: Knockdown of lncRNA PTAR inhibits tumor progression in an orthotopic mouse model of OvCa. a The macroscopic observation of the size and range of metastatic lesions in nude mice. b Representative images of tumors from nude mice bearing xenograft tumors with SKOV3 cells infected with a lentivirus carrying a short hairpin RNA against PTAR (lenti-sh-PTAR) or its scramble negative control (lenti-sh-Scramble). c , d The average number of metastatic nodules and tumor xenograft weights in nude mice. n = 11–13. * P < 0.05. e An immunohistochemistry assay was applied to determine the expression of E-cadherin, fibronectin 1 (FN1), zinc finger E-box binding homeobox 1 (ZEB1) and vimentin in a cell-derived xenograft tumor model. f , g The expression of epithelial-mesenchymal transition (EMT) relevant markers, including E-cadherin, vimentin, FN1 and ZEB1 in tissues derived from xenograft tumors, as determined by qRT-PCR and western blot, respectively. n = 6. * P < 0.05

Article Snippet: The lentiviral constructs of PTAR shRNA were constructed by Biowit Technology (Shenzhen, China).

Techniques: Knockdown, Infection, shRNA, Negative Control, Immunohistochemistry, Expressing, Binding Assay, Derivative Assay, Quantitative RT-PCR, Western Blot

Journal: Molecular Cancer

Article Title: LncRNA PTAR promotes EMT and invasion-metastasis in serous ovarian cancer by competitively binding miR-101-3p to regulate ZEB1 expression

doi: 10.1186/s12943-018-0870-5

Figure Lengend Snippet: PTAR promotes EMT and metastasis through the modulation of miR-101/ZEB1. The SKOV3, A2780 and OVCAR3 cells were transfected with PTAR plasmid with or without miR-101 mimic. a, b Wound healing assays showing cell migration in SKOV3 and A2780 cells. n = 4, * P < 0.05 vs. pcDNA3.1, # P < 0.05 vs. PTAR. c , d Migration assays in SKOV3 and A2780 cells, respectively. e The invasiveness of SKOV3 cells was determined by an invasion assay using matrigel transwell chambers. n = 4, ** p < 0.01. f The protein levels of E-cadherin, vimentin, FN1 and ZEB1 in SKOV3 cells. n = 5, * P < 0.05 vs. pcDNA3.1, # P < 0.05 vs. PTAR. g The protein levels of E-cadherin, vimentin, FN1 and ZEB1 in A2780 cells. n = 5, * P < 0.05 vs. pcDNA3.1, # P < 0.05 vs. PTAR. h The protein levels of E-cadherin and ZEB1 in OVCAR3 cells. n = 3, * P < 0.05 vs. pcDNA3.1, # P < 0.05 vs. PTAR

Article Snippet: The lentiviral constructs of PTAR shRNA were constructed by Biowit Technology (Shenzhen, China).

Techniques: Transfection, Plasmid Preparation, Migration, Invasion Assay

Journal: Molecular Cancer

Article Title: LncRNA PTAR promotes EMT and invasion-metastasis in serous ovarian cancer by competitively binding miR-101-3p to regulate ZEB1 expression

doi: 10.1186/s12943-018-0870-5

Figure Lengend Snippet: Silencing PTAR alleviates ovarian cancer cell tumorigenicity. The SKOV3, A2780 and OVCAR3 cells were transfected with sh-PTAR with or without miR-101 inhibition in the presence or absence of 10 ng/ml TGF-β1 for 48 h. a , b Wound healing assays were performed both in SKOV3 and A2780 cells treated as specified. n = 4, * P < 0.05 vs. Ctrl, # P < 0.05 vs. TGF-β1 + sh-Scram, & P < 0.05 vs. TGF-β1 + sh-PTAR. c , d Cell migration in SKOV3 and A2780 cells was determined using transwell chambers. n = 4, ** P < 0.01. e The invasion ability of SKOV3 cells was evaluated using matrigel transwell chambers. n = 4, ** P < 0.01. f , g The protein levels of E-cadherin, vimentin, FN1 and ZEB1 in SKOV3 and A2780 cells. n = 5, * P < 0.05 vs. Ctrl, # P < 0.05 vs. TGF-β1 + sh-Scram, & P < 0.05 vs. TGF-β1 + sh-PTAR. h The protein levels of E-cadherin and ZEB1 in OVCAR3 cells. n = 3, * P < 0.05 vs. Ctrl, # P < 0.05 vs. TGF-β1 + sh-Scram, & P < 0.05 vs. TGF-β1 + sh-PTAR

Article Snippet: The lentiviral constructs of PTAR shRNA were constructed by Biowit Technology (Shenzhen, China).

Techniques: Transfection, Inhibition, Migration

Journal: Molecular Cancer

Article Title: LncRNA PTAR promotes EMT and invasion-metastasis in serous ovarian cancer by competitively binding miR-101-3p to regulate ZEB1 expression

doi: 10.1186/s12943-018-0870-5

Figure Lengend Snippet: PTAR negatively regulates miR-101 expression and activity in OvCa cells. a , b The expression of PTAR and miR-101 in SKOV3 and A2780 cells after treatment with 10 ng/ml TGF-β1 for 48 h. c , d Analysis with qRT-PCR was used to assess the expression of PTAR and miR-101 in SKOV3 and A2780 cells in response to PTAR overexpression. e The knockdown efficiency of the sh-PTAR plasmid was confirmed by qRT-PCR analysis. f Expression of miR-101 was detected in OvCa cells in response to PTAR suppression. g The luciferase reporter activity of chimeric vectors carrying the luciferase gene and a fragment of PTAR containing the (WT) binding site or a mutated binding site for miR-101. h , i The luciferase activity of miR-101 sensor in OvCa cells transfected as specified. n = 5. * P < 0.05

Article Snippet: The lentiviral constructs of PTAR shRNA were constructed by Biowit Technology (Shenzhen, China).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Over Expression, Knockdown, Plasmid Preparation, Luciferase, Binding Assay, Transfection

Journal: Molecular Cancer

Article Title: LncRNA PTAR promotes EMT and invasion-metastasis in serous ovarian cancer by competitively binding miR-101-3p to regulate ZEB1 expression

doi: 10.1186/s12943-018-0870-5

Figure Lengend Snippet: Overexpression of miR-101 attenuated TGF-β1-induced metastasis in OvCa cell lines. a , b Wound healing assays performed in SKOV3 and A2780 cells, respectively. Images were captured at 0, 24, and 48 h. n = 4, * P < 0.05 vs. Ctrl, # P < 0.05 vs. TGF-β1. c , d The migration ability of SKOV3 and A2780 cells was determined using transwell assays. n = 4, ** P < 0.01. e The expression levels of epithelial and mesenchymal markers were detected by western blot in SKOV3 cells. n = 5, * P < 0.05 vs. Ctrl, # P < 0.05 vs. TGF-β1. f Up-regulation of lncRNA PTAR enhanced ZEB1 expression by competitively binding miR-101, which promoted OvCa cell EMT and invasion

Article Snippet: The lentiviral constructs of PTAR shRNA were constructed by Biowit Technology (Shenzhen, China).

Techniques: Over Expression, Migration, Expressing, Western Blot, Binding Assay