Journal: Cancer Gene Therapy

Article Title: NrCAM secreted by endometrial stromal cells enhances the progestin sensitivity of endometrial cancer cells through epigenetic modulation of PRB

doi: 10.1038/s41417-022-00467-0

Figure Lengend Snippet: A Cluster analysis of MPA-regulated genes encoding secretory proteins in ESCs. ESCs were treated with 10 μM MPA or ethanol (EtOH) for 6 h before RNA sequencing. B mRNA levels of NrCAM , BMP2 , WISP1 , and ITGA10 were significantly upregulated in ESCs after MPA treatment. Silencing PR expression with si PGR in ESCs weakened MPA-induced upregulation of these four proteins. ESCs or ESCs-si PGR were treated with or without 10 μM MPA for 6 h. Eleven candidate gene mRNA levels were reevaluated by real-time PCR. C Exogenous NrCAM inhibited EC cell proliferation in a dose-dependent manner. Ishikawa and ECC-1 cells were treated with 0, 1, 10, 100, and 1000 ng/mL NrCAM for 48 h before CCK-8 assays. D Exogenous NrCAM inhibited EC cell proliferation in a time-dependent manner. Ishikawa and ECC-1 cells were treated with 1000 ng/mL NrCAM for 24, 48, and 72 h before CCK-8 assays. E MPA promoted NrCAM protein expression in ESCs in a dose-dependent manner. NrCAM expression was detected by western blotting. ESCs were treated with 0, 5, 10, and 20 μM MPA for 48 h. F MPA promoted NrCAM secretion in ESCs by ELISA. ESCs were treated with MPA at the indicated dose for 48 h (left) or 10 μM MPA for 24, 48, or 72 h (right). The CM extracted from ESCs was collected to measure NrCAM concentration by ELISA. G MPA-induced NrCAM protein expression was attenuated by silencing PGR in ESCs. ESCs or ESCs-si PGR were treated with 10 μM MPA for 48 h before western blotting analysis. H NrCAM and MPA cotreatment had a stronger inhibitory effect on EC cell proliferation than MPA or NrCAM alone. Ishikawa and ECC-1 cells were treated with 1000 ng/mL NrCAM and/or 10 μM MPA for 48 h before CCK-8 assays. I Transfection efficiency of siRNAs targeting NrCAM was confirmed by real-time PCR and western blotting. J The inhibitory effect of ESCs on EC cell proliferation was blocked by silencing NrCAM expression in ESCs. After transfection with si NrCAM or siCtrl for 8 h, ESCs were treated with or without 10 μM MPA for 48 h, then for CM collection. Ishikawa and ECC-1 cells were treated with 10 μM MPA, CM (ESCs-si NrCAM -2) and CM (ESCs-si NrCAM -2 + MPA) for 48 h before CCK-8 assays. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s. not significant.

Article Snippet: Drugs used in this study included MPA (Sigma-Aldrich, St. Louis, MO, USA), recombinant human NrCAM protein (CC04, NovoProtein, Shanghai, China), recombinant human BMP2 protein (C012, NovoProtein), recombinant human WISP1 protein (10442-H08H, Sino Biological, Beijing, China), and recombinant human ITGA10 protein (5895-AB-050, R&D Systems, Minneapolis, MN, USA) at the indicated doses for the indicated periods.

Techniques: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, CCK-8 Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: WISP1 expression is increased in melanoma and is associated with reduced overall survival of patients diagnosed with primary melanoma. A, Comparison of WISP1 mRNA expression in benign skin conditions (normal skin and benign melanocytic skin nevus) to primary melanoma. Original expression profiles were from . P-values calculated using ANOVA with post-hoc Tukey HSD test. B, Representative original and deconvoluted color images derived from human normal skin and melanoma tissue microarray probed using a WISP1 antibody (HPA007121) and imaged using 3,3’ diaminobenzidine and stained using hematoxylin for a normal skin (left) and two melanoma (right) tissue samples (original tissue microarray images were obtained from www.proteinatlas.org ) . Deconvoluted intensity of WISP1 staining is shown in red while cellular structures stained using hematoxylin are shown in blue. Arrows indicate melanocytes in epidermis and arrowheads indicate fibroblasts in dermis (stroma). C, The average WISP1 staining within normal skin and primary melanoma tissue samples. D, Distributions in non-zero pixel intensity values of WISP1 staining for normal skin (black curves) and primary melanoma (red curves) tissue samples. Numbers indicate the percentage of the distribution that have normalized pixel intensity values greater than 0.2. E, Kaplan-Meier estimate of overall survival of patients diagnosed with primary melanoma stratified by WISP1 transcript abundance (data from TCGA). Sample numbers and p-values calculated using the Peto & Peto modification of the Gehan-Wilcoxon test are indicated. F, Patient population characteristics of WISP1 high and WISP1 low groups. Statistical differences among categorical data and age were assessed using Fisher’s Exact test and Student’s t-Test, respectively (n.s. indicates p-value > 0.05).

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Expressing, Comparison, Derivative Assay, Microarray, Staining, Modification

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: WISP1 knockout in mouse and human melanoma cells inhibited tumor cell migration and invasion. A, 48-hour 2D growth of mouse metastatic melanoma cell line B16F10 and two B16F10 Wisp1-knockout cells (-KO1 and -KO2). B, Anchorage-independent growth assay of B16F10 and the two knockout cells in soft agar. Colonies were fixed and counted after 14 days. A representative staining image for each sample is shown on left, colony counts is plotted on the right. C, Wound healing assay of B16F10 and the two knockout cells. Scratches were created on 6-well plates in biological triplicate and the healing rate was calculated after 24 hours. D, Boyden transwell migration assay of B16F10 and the two knockout cells. A representative staining image for each sample is shown on left, relative migration efficiency is graphed on the right. E, Boyden transwell invasion assay of B16F10 and the two knockout cells. F, Boyden transwell invasion assay of human metastatic melanoma cell line RPMI-7951 and its two Wisp1-knockout cells (-KO1 and -KO2). G, Transwell migration assay of B16F10 and its knockout cell (-KO1) using conditioned media with different concentration of Wisp1 as chemoattractant. B16F10 migrated cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative migration efficiency and compared with other cells. H, Transwell invasion assay of B16F10 and the two knockout cells using conditioned media with different concentration of Wisp1 as chemoattractant. B16F10 invaded cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative invasion efficiency and compared with other cells. Statistical significance was determined by Student’s t test, where a p-value < 0.05 was considered significant and asterisks was used to indicate calculated range in p-values. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; and ns: not significant.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Migration, Growth Assay, Staining, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay, Concentration Assay

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Real time genomic qPCR revealed that Wisp1 knockout repressed the spontaneous metastasis of melanoma cell line B16F10 in C57BL/6Ncrl mice. Growth of tumors derived from B16F10 or its knockout cell (-KO2) were monitored following subcutaneous injection in NSG mice (A: B16F10 (n = 5) or WISP1 KO cell (n = 5)) or in C57BL/6Ncrl mice (B: B16F10 (n=6) or WISP1 KO cell (n=6)). After 21 days, remaining C57BL/6crl mice (n = 4 in each group) were euthanized and lungs and livers were assayed for B16F10 tumor cells using real time genomic qPCR, as described in Materials and Methods. (C) Representative lungs and livers from C57BL/6Ncrl mice with B16F10 or knockout cell at day 21. (D) Real time genomic qPCR results showed quantitative tumor lung and liver metastatic burden in spontaneous metastasis assays, n.d., not detected.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Derivative Assay, Injection

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Wisp1 knockout repressed the experimental metastasis of melanoma cell line B16F10 in immunodeficient NSG mice and immunocompetent C57BL/6Ncrl mice. Experimental metastasis assays were performed in NSG mice ( A-F ) and C57BL/6Ncrl mice ( G-I ) using B16F10 and indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (N=5) and three representative images were shown. These experiments were repeated and similar results were achieved. A, Bioluminescence imaging performed one day before NSG mice were euthanized. All animals were compared with the same bioluminescence scale. B-C, Tumor lung metastases (black colonies) of NSG mice as captured by photography ( B ) and real time genomic qPCR ( C ). Quantitative tumor lung metastatic burden was assayed and presented as tumor cell number within 10,000 mouse tissue cells. D-E , Tumor liver metastases (black and white nodules) of NSG mice as captured by photography ( D ) and real time genomic qPCR ( E ). Quantitative tumor liver metastatic burden was assayed and presented as tumor cell number within 10,000 mouse tissue cells. F, Tumor kidney metastases (black colonies) of NSG mice as captured by photography. G, Bioluminescence imaging performed one day before C57BL/6Ncrl mice were euthanized. All animals were compared with the same bioluminescence scale. H-I, Tumor lung metastases of C57BL/6Ncrl mice as captured by photography ( H ) and real time genomic qPCR ( I ). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Injection, Imaging

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Wisp1 knockout repressed the experimental metastasis of melanoma cell line YUMM1.7 in NSG and C57BL/6Ncrl mice. Experimental metastasis assays were performed in NSG ( A-D ) and C57BL/6Ncrl ( E-H ) mice using YUMM1.7 and indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (N=5) and two representative images were shown. A, Bioluminescence imaging performed one day before NSG mice were euthanized. All animals were compared with the same bioluminescence scale. B, Tumor lung metastases (white nodules) of NSG mice as captured by photography. C, Real time genomic qPCR quantitatively comparing tumor lung metastatic burdens (tumor cell number within 10,000 mouse tissue cells). D, The whole-body metastasis of tumor cells in NSG mice were plotted and compared using bioluminescence intensity detected in panel (A). Total flux is presented as photon/second (p/s). E, Bioluminescence imaging performed one day before C57BL/6Ncrl mice were euthanized. All animals were compared with the same bioluminescence scale. F, Tumor lung metastases (white nodules) of C57BL/6Ncrl mice as captured by photography. G, Real time genomic qPCR quantitatively comparing tumor lung metastatic burdens. H, The whole-body metastasis of tumor cells in C57BL/6Ncrl mice were plotted and compared using bioluminescence intensity detected in panel (E). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Injection, Imaging

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: WISP1 induced an EMT gene signature in mouse/human melanoma cells. Unless otherwise specified, all cells were plated on 6-well plates in complete growth medium for 48 hours before harvested for RNA analysis or treated with indicated conditioned medium or recombinant protein. A, mRNA expression, revealed by real-time quantitative RT-PCR, of select EMT marker genes and Mitf in uninvaded and invaded B16F10 cells from Boyden transwell invasion assay. B, Immunoblot analysis of Wisp1 protein to confirm the disruption of Wisp1 gene in B16F10 and YUMM1.7 knockout cells. 20μg of whole cells lysate was load in each lane and P-actin was used as internal loading control. B16F10-KO1-mWisp1 cell, in which mouse Wisp1 expression was resumed with retroviral transduction, was used as a positive control. C, Immunoblot analysis of certain EMT marker proteins in B16F10 and YUMM1.7 knockout cells. 20μg of whole cells lysate was load in each lane and all cells were compared on the same gel to reveal the relative intensity of each protein. D, Comparison of EMT marker gene expression in mouse melanoma B16F10 and its two Wisp1-knockout cells (-KO1 and -KO2). E Comparison of EMT marker gene expression in mouse melanoma YUMM1.7 and its two Wisp1-knockout cells (-KO1 and - KO2). F, Comparison of EMT marker gene expression in human melanoma RPMI-7951 and its two Wisp1-knockout cells (-KO1 and -KO2). G, Stimulation of EMT marker gene expression with recombinant mouse Wisp1 protein (rmWisp1). B16F10-KO1 cells were treated with rmWisp1 (final 5μg/ml) and harvested at indicated time point for real-time quantitative RT-PCR analysis. H, Stimulation of EMT marker gene expression with Wisp1-overexpressed or Wisp1-immunodepleted conditioned medium. The conditioned media were pre-treated with indicated antibodies for 30 minutes before used on Wisp1-knockout B16F10 cells (-KO1). The cells were collected for real-time qRT-PCR after 3 hour treatment. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Marker, Transwell Invasion Assay, Western Blot, Disruption, Knock-Out, Transduction, Positive Control, Comparison

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Snai1 overexpression in B16F10 Wisp1-knockout cell rescued the repression on tumor invasion in vitro and metastasis in vivo. A, Immunoblot analysis of Wisp1 and Snai1 using B16F10-KO1 cell that were transduced with retroviral vector control (-pBabe), or retrovirus expressing either mouse Wisp1 (-mWisp1) or human Snai1 (-hSnai1). B, Comparison of EMT marker gene expression after overexpression of Snai1 or reintroduction of Wisp1 in B16F10-KO1 cells. Cells were plated on 6-well plates in complete growth medium for 48 hours before harvested for RNA analysis. C, Boyden transwell invasion assay after overexpression of Snai1 or reintroduction of Wisp1 in B16F10-KO1 cells. A representative staining image for each sample is shown on left, relative invasion efficiency is graphed on the right. D, Experimental metastasis assay in NSG mice using indicated cells. Each group contained 3-4 mice. All mice were imaged one day before the end of the assay and representative bioluminescence images were shown. E, Representative lung and liver images from NSG mice in experimental metastasis assay described in panel ( D ). Metastatic tumor colonies on lung surface from mice with (-mWisp1) or (-hSnai1) cells were pointed by arrows. F, Real time genomic qPCR for lungs and livers from experimental metastasis assay in panel ( D ). The quantitative tumor metastatic burdens were presented as tumor cell number within 10,000 mouse tissue cells. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Over Expression, Knock-Out, In Vitro, In Vivo, Western Blot, Transduction, Plasmid Preparation, Expressing, Comparison, Marker, Transwell Invasion Assay, Staining

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Wisp1 activated Akt/MAPK signaling and promoted EMT marker gene expression in mouse melanoma cells. Unless otherwise specified, cell treatment for kinase immunoblot analysis maintained for 30 minutes before cells were lysed for protein extraction while cells were treated for 3 hours prior to RNA extraction for comparing EMT marker gene expression. A, EMT marker gene expression after inhibiting Akt and/or MAPK signaling in B16F10 cells. Cells were treated with specific phospho-Akt inhibitor MK-2206 and/or phospho-MAPK inhibitor U0126. Immunoblot for phospho-Akt and phospho-Erk1/2 inhibition was shown on the right upper corner. Pan-Akt and total Erk1/2 were also probed as loading control. B, EMT marker gene expression after inhibiting Akt and/or MAPK signaling in YUMM1.7 cells, with DMSO as control. C, Immunoblot for phospho-Akt and phospho-Erk1/2 in indicated mouse melanoma cells with treatment of recombinant mouse Wisp1 protein (rmWisp1, final 5μg/ml). Cells grown on 6-well plates in complete DMEM for 48 hours and serum-free DMEM (SFM) for another 48 hours before rmWISP1 was added. Pan-Akt and total Erk1/2 were probed as loading control. D, Immunoblot analysis of Akt/MAPK activation in B16F10 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were grown on 6-well plates in complete DMEM for 48 hours (0 hour point for SFM) and switched to SFM for 24 hour or 48 hours. Indicated cells were treated with rmWisp1 for 30 minutes following 0, 24, or 48 hours in SFM before analyzed for kinase activation. The first lane loaded with YUMM 1.7 at 0 hour to compare the relative kinase level between B16F10 and YUMM1.7 cells. E, Immunoblot for Akt/MAPK activation in YUMM1.7 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were treated similarly as described in panel (D). The first lane loaded with B16F10 at 0 hour to compare the relative kinase level between B16F10 and YUMM1.7 cells. F Snai1 activation and E-cadherin repression in B16F10 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were treated similarly as described in panel (D) except that rmWisp1 treatment at each point maintained for 3 hours. G-H, EMT marker gene expression after Akt/MAPK activation in B16F10-KO1 (G) or YUMM1.7-KO1 (H) by rmWisp1 was blocked by Akt/MAPK inhibitors. rmWisp1 with DMSO or inhibitors was added after indicated cells were grown on 6-well plates in complete DMEM for 48 hours and in SFM for 24 hours. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Marker, Expressing, Western Blot, Protein Extraction, RNA Extraction, Inhibition, Recombinant, Activation Assay, Knock-Out

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: Digital cytometry deconvolutes a bulk transcriptomic profile using gene signatures that correspond to different stromal, malignant, and immune cell types. The results estimate the prevalence of the different cell types within the tissue sample, that is the digital cytometry features. By using bulk transcriptomic profiles of defined patient populations, underlying variation in the inferred cellular composition coupled with features associated with a patient sample, such as over-expression of a secreted gene product by malignant cells, can be used to estimate how the heterocellular network is impacted by a genetic alteration intrinsic to the malignant cell using Bayesian Network inference. To illustrate the approach, we focused on malignant cell expression of Cell Communication Network factor 4 (CCN4), a secreted matricellular protein. The resulting directed acyclic graph represents the collective conditional independence among the modeled features, or nodes, of the network.

Article Snippet: The concentration of CCN4 protein in the cell culture media from those wells was assayed using the Human WISP-1/CCN4 DuoSet ELISA Kit (R&D Systems, Minneapolis, MN) to confirm CCN4 knockout.

Techniques: Cytometry, Over Expression, Expressing

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: The nodes of the graph represent features, such as CCN4 gene expression (rectangle), sample attribute (hexagon), or the prevalence of a particular cell type/state (oval). The edges represent inferred causal relationships among the nodes. The black lines with arrow heads represent a positive causal relation while red lines with horizontal bars represent a negative or inhibitory causal relation, where the extent of influence of the parental node is annotated by the number beside the edge. The number included within the node symbol represents the average normalized value of the digital cytometry feature within the dataset with values of all of the parental nodes set to zero. The width of the edge is proportional to the posterior probability of inclusion into the DAG.

Article Snippet: The concentration of CCN4 protein in the cell culture media from those wells was assayed using the Human WISP-1/CCN4 DuoSet ELISA Kit (R&D Systems, Minneapolis, MN) to confirm CCN4 knockout.

Techniques: Expressing, Cytometry

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A The percentage of live CD45+ cells isolated from tumors generated by inoculating s.c. with WT (red) and CCN4 KO (blue) variants of B16F0 (o and x’s) and YUMM1.7 (□ and +’s) cells, where the log-linear trends are highlighted by dotted lines. CD45+ values were obtained from three different antibody panels that quantified T cells, B/NK cells, and myeloid cells in TIL isolates from each mouse. B A comparison of the ratio of NK cells (black), CD8+ T cells (red), CD4+ T cells (blue), and B cells (green) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). C The difference in the mean prevalence of the infiltrating immune cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry from the BRCA (dark gray) and SKCM (light gray) datasets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. D TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by NK cells, CD8+ T cells, CD4+ T cells, and B cells (top to bottom) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO pairs using two-sided Student’s t test.

Article Snippet: The concentration of CCN4 protein in the cell culture media from those wells was assayed using the Human WISP-1/CCN4 DuoSet ELISA Kit (R&D Systems, Minneapolis, MN) to confirm CCN4 knockout.

Techniques: Isolation, Generated, Comparison, Cytometry

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A A comparison of the ratio of CD11c- (black) and CD11c+ (gray) macrophages, Dendritic cells (yellow), CD11c+ MDSC (green), MDSC (blue), and Neutrophils (red) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). B The difference in prevalence of the myeloid cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry of the BRCA (dark gray) and SKCM (light gray) data sets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. Macrophages are the only myeloid cell subset inferred from the BRCA and SKCM datasets and are assumed to be related to CD11c+ macrophages in mouse models. C A representative scatter plot of GR1 versus CD11c expression in gated live CD45+ CD11b+ TILs obtained from WT (top) and CCN4 KO (bottom) YUMM1.7 tumors. D – F TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by myeloid-derived suppressor cell subsets ( D : MDSC (top) and CD11c+ MDSC (bottom)) and other myeloid cell subsets ( E : CD11c- (top) and CD11c+ (bottom) macrophages, F : neutrophils (top) and dendritic cells (bottom)) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO variants using two-sided Student’s t test.

Article Snippet: The concentration of CCN4 protein in the cell culture media from those wells was assayed using the Human WISP-1/CCN4 DuoSet ELISA Kit (R&D Systems, Minneapolis, MN) to confirm CCN4 knockout.

Techniques: Comparison, Generated, Cytometry, Expressing, Derivative Assay

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A Expression of genes for transcription factors (left panel - Snai1: red triangle, Snai2: blue diamond, Zeb1: black circle, and Zeb2: gray square) and adhesion proteins (right panel - Cdh1: blue triangle, Cdh2: black circle, Fn1: red square) associated with the epithelial-mesenchymal transition were assayed as a function of time following addition of rmCCN4 to CCN4 KO YUMM1.7 (top row) and CCN4 KO B16F0 cells (bottom row). Colored asterisks indicate whether gene at a particular time point was significantly different than untreated cells, where n = 3 biological independent samples. B The distribution in cell trace staining among live CD4 + (left panel) and CD8 + (right panel) T cells stimulated with α CD3/ α CD28 (AP beads) alone or in the presence of media conditioned by WT B16F0 cells (AP beads + WT TCM), media conditioned by CCN4 KO B16F0 cells (AP beads + CCN4 KO TCM), or with 10 ng/ml of recombinant mouse CCN4 (AP beads + rCCN4). The distribution in the corresponding unstimulated cells (gray) are shown at the bottom. The colored vertical lines indicate the predicted dilution of cell trace staining in each generation based on the unstimulated controls. C Bivariate projection of the weights of genes within the resting (y-axis) and activated (x-axis) NK cell signatures. D Using spleens from C57BL/6 mice that were challenged with YUMM1.7 cells, isolated CD8+ T cells were assayed by in vitro ELISpot for IFN γ expression using variants of the YUMM1.7 cell line as targets (CCN4 KO YUMM1.7 with a blank inducible expression vector and CCN4 KO YUMM1.7 with a CCN4 inducible expression vector). To induce CCN4 expression, these YUMM1.7 variants were also cultured in the absence (−) or presence of doxycycline (+) and quantified following 24 h co-culture. Statistical significance between WT and CCN4 KO variants was assessed using two-way ANOVA followed by Tukey’s multiple comparison ad hoc post-test, where n = 6 biologically independent samples. Results summarized as mean ± s.d.

Article Snippet: The concentration of CCN4 protein in the cell culture media from those wells was assayed using the Human WISP-1/CCN4 DuoSet ELISA Kit (R&D Systems, Minneapolis, MN) to confirm CCN4 knockout.

Techniques: Expressing, Staining, Recombinant, Isolation, In Vitro, Enzyme-linked Immunospot, Plasmid Preparation, Cell Culture, Co-Culture Assay, Comparison