Journal: Journal of Cancer

Article Title: MYH9 Promotes Growth and Metastasis via Activation of MAPK/AKT Signaling in Colorectal Cancer

doi: 10.7150/jca.27635

Figure Lengend Snippet: MYH9 is upregulated in CRC tissues compared with normal mucosa. A, MYH9 expression in CRC samples and paired non-neoplastic tissues was detected by western blot. β-tubulin was selected as a control for normalization. B, MYH9 staining intensity defined into four groups as negative, weak, moderate and strong in clinical CRC samples and adjacent normal tissues by IHC. C, MYH9 expression in the tumors and normal samples was examined by IHC. Representative figures are shown. Scale bars represent 50 µm. N, normal tissues. T, tumor tissues. The raw blots/gels are presented in Supplementary Figure .

Article Snippet: A stable lentiviral vector with overexpression of MYH9 was also constructed with human full-length MYH9 cDNA (GeneCopoeia, Rockville, USA).

Techniques: Expressing, Western Blot, Control, Staining

Journal: Journal of Cancer

Article Title: MYH9 Promotes Growth and Metastasis via Activation of MAPK/AKT Signaling in Colorectal Cancer

doi: 10.7150/jca.27635

Figure Lengend Snippet: MYH9 is positively correlated with progression and poor survival in CRC. A, Kaplan-Meier survival curves and univariate analyses (log-rank) for CRC patients with low expression (< median, n=97) and high expression (≥median, n=99) of MYH9 in the testing cohorts derived from a published high-throughput microarray dataset (NCBI/GEO/GSE87211; n=203). B, Kaplan-Meier plot of overall survival of 100 CRC patients (log rank=10.390, P =0.001). C, Overall survival of 38 CRC patients with N1 and N2 stage (log rank=5.320, P =0.021). D, Overall survival of 62 CRC patients with N0 stage (log rank=3.174, P =0.075).

Article Snippet: A stable lentiviral vector with overexpression of MYH9 was also constructed with human full-length MYH9 cDNA (GeneCopoeia, Rockville, USA).

Techniques: Expressing, Derivative Assay, High Throughput Screening Assay, Microarray

Journal: Journal of Cancer

Article Title: MYH9 Promotes Growth and Metastasis via Activation of MAPK/AKT Signaling in Colorectal Cancer

doi: 10.7150/jca.27635

Figure Lengend Snippet: Endogenous overexpression of MYH9 accelerates CRC cell growth and metastasis in vitro and in vivo . A, MYH9 expression level in 6 CRC cell lines were explored by western blot (left panel) and quantified (right panel). B, MYH9 mRNA expression (left panel) and protein expression (right panel) in SW620 cells infected with lentivirus including human full-length MYH9 cDNA (LV-MYH9) and empty vector (LV-Con). C, CCK-8 (left panel) and plate colony formation assay (right panel) in indicated cells. D, Cell migration (left panel) and invasion assay (right panel) in indicated cells. E, Nude mice tumorigenicity assay using indicated cells injected into hind limb flank. Tumor size and weight were shown in the right. F. H&E and MYH9 along with Ki67 staining of the tumors collected from the nude mice. Scale bars represent 50 µm. Error bars represented mean ± SD. Three independent experiments were carried out for each assay. (*), P <0.05; (**), P <0.01. The raw blots/gels are presented in Supplementary Figure .

Article Snippet: A stable lentiviral vector with overexpression of MYH9 was also constructed with human full-length MYH9 cDNA (GeneCopoeia, Rockville, USA).

Techniques: Over Expression, In Vitro, In Vivo, Expressing, Western Blot, Infection, Plasmid Preparation, CCK-8 Assay, Colony Assay, Migration, Invasion Assay, Tumorigenicity Assay, Injection, Staining

Journal: Journal of Cancer

Article Title: MYH9 Promotes Growth and Metastasis via Activation of MAPK/AKT Signaling in Colorectal Cancer

doi: 10.7150/jca.27635

Figure Lengend Snippet: Silencing of MYH9 inhibits proliferation and migration of CRC cells in vitro and in vivo . A, Knockdown effectiveness of four siRNAs were tested by real-time PCR (left panel) and western blot (right panel). B, Knockdown effectiveness of lentivirus (LV-siMYH9), which was derived from siMYH9-4 was validated by western blot. C, CCK-8 assay in indicated cells. D, Colony formation, migration and invasion assays in indicated cells. E, In vivo tumor formation assay using nude mice with tumor size and weight in the right. F, Representative figures of the tumors developed in nude mice with H&E, MYH9 and Ki67 staining. Scale bars represent 50 µm. Error bars served as mean ± SD for three independent experiments. Each experiment was repeated for three times. (*), P <0.05; (**), P <0.01. The raw blots/gels are presented in Supplementary Figure .

Article Snippet: A stable lentiviral vector with overexpression of MYH9 was also constructed with human full-length MYH9 cDNA (GeneCopoeia, Rockville, USA).

Techniques: Migration, In Vitro, In Vivo, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay, CCK-8 Assay, Tube Formation Assay, Staining

Journal: Journal of Cancer

Article Title: MYH9 Promotes Growth and Metastasis via Activation of MAPK/AKT Signaling in Colorectal Cancer

doi: 10.7150/jca.27635

Figure Lengend Snippet: EMT is required for MYH9-mediated CRC cell aggressive phenotypes. A, Cell morphology under inverted microscope in indicated cells (scale bars represent 20 µm). B, Western blot of EMT markers and MMP9 in indicated cells. Immunosignals of different protein expression level were quantified by greyscale scanning software (Quantity one) and relative protein abundance was normalized by β-tubulin. C, Immunofluorescence of EMT markers ZO-1 and β-catenin in indicated cells (scale bars represent 10 µm). D, EMT markers ZO-1 and β-catenin expression in nude mice derived tumors with MYH9 overexpression and knockdown by IHC. Scale bars represent 20 µm. The raw blots/gels are presented in Supplementary Figure .

Article Snippet: A stable lentiviral vector with overexpression of MYH9 was also constructed with human full-length MYH9 cDNA (GeneCopoeia, Rockville, USA).

Techniques: Inverted Microscopy, Western Blot, Expressing, Software, Quantitative Proteomics, Immunofluorescence, Derivative Assay, Over Expression, Knockdown

Journal: Journal of Cancer

Article Title: MYH9 Promotes Growth and Metastasis via Activation of MAPK/AKT Signaling in Colorectal Cancer

doi: 10.7150/jca.27635

Figure Lengend Snippet: MYH9 induces EMT and promotes CRC progression through MAPK and AKT signaling . A, Western blot analysis of protein expression related to AKT/ERK signaling pathway. B, Western blot analysis of AKT/ERK signaling pathway and EMT markers in indicated cells treated with AKT inhibitor LY29004 and MEK inhibitor U0126. Immunosignals of different protein expression level were quantified by greyscale scanning software (Quantity one) and relative protein abundance was normalized by β-tubulin. C, Transwell assay in indicated cells. D, Cell morphology of indicated cells was observed under inverted microscope. Scale bars represent 50 µm. Error bars represented mean ± SD from three independent experiments. (*), P <0.05; (**), P <0.01. The raw blots/gels are presented in Supplementary Figure &6.

Article Snippet: A stable lentiviral vector with overexpression of MYH9 was also constructed with human full-length MYH9 cDNA (GeneCopoeia, Rockville, USA).

Techniques: Western Blot, Expressing, Software, Quantitative Proteomics, Transwell Assay, Inverted Microscopy

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet: Scoring of IHC-stained patient-derived samples.

Article Snippet: TaqMan human TNC (Hs01115665_m1) , Thermo Fisher , Cat #: 4453320.

Techniques:

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet: (A) Schematic of the workflow of isolating, culturing, and comparing the transcriptome of brain metastatic (BrM) derivatives to the parental breast cancer cell lines they were isolated from. (B) GSEA showing that, in comparison to corresponding parental cell lines, the ECM organization pathway is enriched in the brain metastatic derivatives of HCC1954 and MMTV-ErbB2 HER2BC cells and not in MDA231 TNBC cells. Color shades indicate BH-adjusted P values of normalized enrichment scores. (C) Z-scored expression of the matrisome genes TNC , COL4A1 , IL24 , and SEMA7A that are differentially upregulated in HCC1954 and MMTV-Erbb2 HER2BC brain metastatic derivatives (BrM) in reference to their corresponding parental (Par) cell lines. (D) Forest plots showing the hazard ratio of the expression of matrisome genes COL4A1 , CST6 , SEMA7A , TNC , IL24 , and S100A7A for relapse-free survival of HER2BC breast cancer patients. Gene names in red indicate core matrisome genes. P values from top to bottom are as follows: 0.0012, 0.0135, 0.0894, 0.0641, 0.1649, 0.0889. (E) Schematic of the MetMap workflow using barcoded cancer cell line pools for high-throughput metastatic potential profiling (adapted from Ref. ). Relative metastatic potential was quantified by deep sequencing of barcode abundance from tissue. Comparing the transcriptome of in vivo brain metastases to that of in vitro cell culture per multiplexed cell line pool yielded the log2 fold change (log2FC) of gene expression shown in (F). (F) Relative in vivo expression, visualized by the log2FC values shown in a descending order, of the top ECM component genes that are differentially upregulated in the brain metastasis samples composed predominantly of HER2BC cells (pink) than of TNBC cells (blue) (see for statistical analysis of the association between relative in vivo expression and percent of HER2BC cells across multiplexed brain metastasis samples). (G) GSEA showing that ECM-related pathways are enriched in multiplexed brain metastasis samples composed predominantly of HER2BC cells (denoted in pink in (F)) than of TNBC cells (in blue in (F)). Top four positively enriched gene sets are Gene Ontology (GO) terms of 1, collagen containing extracellular matrix (GO:0062023), 2, extracellular matrix (GO:0031012), 3, extracellular structure organization (GO:0043062), and 4, extracellular matrix structural constituent (GO:0005201).

Article Snippet: TaqMan human TNC (Hs01115665_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Isolation, Comparison, Expressing, High Throughput Screening Assay, Sequencing, In Vivo, In Vitro, Cell Culture, Gene Expression

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet: (A) Representative IF staining showing the TNC deposition in incipient spheroidal colonies formed by HCC1954-BrM cells 7 days post-intracardiac inoculation. Scale bar, 50 μm. (B) Quantification of the IF staining signal of tenascin C (TNC), fibronectin (FN), and periostin (POSTN) per GFP+ (tumor) unit area in HCC1954-BrM HER2BC and MDA231-BrM TNBC brain metastatic colonies. RFU, relative fluorescence unit. Mean ± SEM. (C) (Left panel) Quantification and (right panel) representative IHC staining and associated TNC level scores (corresponding H&E staining shown in ) of TNC in brain metastasis tissue samples derived from TNBC patients (n = 13) and HER2BC patients (n = 18). Scale bars, 200 μm.

Article Snippet: TaqMan human TNC (Hs01115665_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Staining, Fluorescence, Immunohistochemistry, Derivative Assay

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet: (A-B) Effect of the suppression of COL4A1 and TNC expression on oncosphere formation (A) by shRNA in HCC1954-BrM cells and (B) by CRISPRi (2 sgRNAS per target gene) in MMTV-Erbb2-BrM cells, measured by the size of colonies after 5 days of growth in vitro . n = 100–175 colonies/group. Mean ± SEM. (C-D) Effect of the suppression of COL4A1 and TNC expression on brain colonization (C) by shRNA in HCC1954-BrM cells (n = 5 mice/group, 4 weeks after intracardiac inoculation into athymic mice, 2 shRNAs per target gene) and (D) by CRISPRi in MMTV-Erbb2-BrM cells (n = 5–9 mice/group, 3 weeks post-intracardiac inoculation into FVB/NJ mice, 2 sgRNAs per target gene), both quantified by whole-body BLI. (E-G) (E, G) Quantification and (F) representative IF staining of brain metastatic colonies formed by HCC1954-BrM cells expressing either a control vector or shRNAs that deplete COL4A1 or TNC expression. n = 3 mice/group. Mean ± SEM. Unpaired t test. (G) The percent of vascular coopting cells and (E) the number of colonies were determined 1 and 3 weeks post-intracardiac inoculation, respectively. (H) Schematic illustrates distinctive modes of colonization and stromal interface, various cancer cell-intrinsic mediators of colonization, and the induction of distinct DAM responses during the initiation of brain colonization in TNBC and HER2BC. Copyright © 2023 Memorial Sloan Kettering.

Article Snippet: TaqMan human TNC (Hs01115665_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Expressing, shRNA, In Vitro, Staining, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet:

Article Snippet: TaqMan human TNC (Hs01115665_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Blocking Assay, Virus, Derivative Assay, Recombinant, Modification, Saline, Electron Microscopy, cDNA Synthesis, Sample Prep, Microarray, Gene Expression, Synthesized, Plasmid Preparation, Software, Magnetic Beads

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet:

Article Snippet: TaqMan human TNC (Hs01115665_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: shRNA, Plasmid Preparation, Selection, Marker, Retroviral

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet:

Article Snippet: TaqMan human TNC (Hs01115665_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Sequencing