Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: a The expression of α-SMA in tumor stroma was assayed by immunohistochemical staining, indicating that CAFs were abundant in pancreatic tumor stroma. b The morphological images of NAFs and CAFs. c Immunofluorescence staining showed the subcellular localization and the expression of α-SMA and FAP in NAFs and CAFs. Scale bar = 50 μm, magnification, ×400. d The mRNA expression levels of α-SMA, FAP, and FSP1 in NAFs and CAFs (passage 4) isolated from three patients were detected by qRT-PCR analysis. n = 3 (replicating from three patients), *** p < 0.001. e Western blot analysis shows the expression of α-SMA and FAP in NAFs and CAFs derived from four pairs of non-neoplastic pancreatic tissues and tumor tissues. f , g qRT-PCR and western blot analysis show the different expression levels of α-SMA and FAP in CAFs at different passages. Compared with the fourth-passage CAFs (CAFs-P4), the expression of α-SMA and FAP in CAFs-P8 had no significant difference, but the expression in CAFs-P12 significantly decreased. n = 3 (replicating from three patients), ns: not significantly different, * p < 0.05

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence, Isolation, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: a , b The qRT-PCR and western blot analysis show the mRNA and protein levels of SATB-1 in SW1990 and PANC-1 cells cultured with (co-culture) or without CAFs (monoculture). n = 3, *** p < 0.001. c The qRT-PCR analysis shows the mRNA expression of SATB-1 in 32 pancreatic tumor tissues and matched non-neoplastic pancreatic tissues. Dots represent each patient, and error bars indicate standard deviation (SD). n = 3, *** p < 0.001. d Western blot analysis shows the protein levels of SATB-1 in 12 pancreatic cancer tissues (PC) and matched non-neoplastic pancreatic tissues (NP). e , f The qRT-PCR and western blot analyses show the mRNA and protein levels of SATB-1 in various pancreatic cancer cell lines. n = 3

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Quantitative RT-PCR, Western Blot, Cell Culture, Co-Culture Assay, Expressing, Standard Deviation

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: a Wound-healing assay shows the abilities of SW1990 and PANC-1 cells with SATB-1 silenced or co-cultured. b The transwell assay shows the fractions of migrated and invaded SW1990 and PANC-1 cells with SATB-1 silenced or co-cultured. Knockdown of SATB-1 inhibited the migration and invasion abilities of PANC-1 and SW1990 cells. Coculturing with CAFs enhanced the migration and invasion abilities of PANC-1 and SW1990 cells, but neutralization with anti-SDF-1 antibody reduced these abilities. Compared with control pancreatic cancer cells, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. c Wound-healing and transwell assays show the upregulated migration and invasion abilities of Capan-2 and BXPC-3 cells transfected with pcDNA3.1-SATB-1. Compared with control pancreatic cancer cells, n = 3, ** p < 0.01, *** p < 0.001

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Wound Healing Assay, Cell Culture, Transwell Assay, Knockdown, Migration, Neutralization, Control, Transfection

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: The relationship between SDF-1 expression and SATB-1 expression in pancreatic cancer tissues

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-6 promotes pancreatic cancer cell migration by rapidly activating the small GTPase CDC42

doi: 10.1074/jbc.RA118.003276

Figure Lengend Snippet: IL-6 promotes migration of pancreatic cancer cells. A, PANC-1 cells were plated in a chemotactic transwell migration assay in the presence or absence of IL-6 (0–100 ng/ml) for 7 h. PDGF (30 ng/ml) was used as a positive control. As a specificity control, cells were incubated with blocking antibodies to IL-6 receptor (IL6R) or the coreceptor gp130, compared with IgG control, in the presence of IL-6 (50 ng/ml). The percentage of cells that migrated across a transwell filter is shown. Graphed data represent the mean of three to five independent experiments. Error bars represent S.E. * indicates p < 0.05. B, migration of PANC-1 cells was measured by a wound healing assay. Representative images show PANC-1 cells before and after wounding a confluent monolayer in the absence of serum (left) or presence of 10% FBS (right) ±IL-6 (50 ng/ml). White lines indicate the starting (t = 0 h) and ending edges of the migrating cells (t = 24 h for no serum and 16 h for 10% FBS). Graphed data indicate the relative distance migrated, normalized to control cells. C, IL-6 induces filopodial actin protrusions. PANC-1 cells were transfected to express GFP-actin and then imaged using live-cell fluorescence microscopy while stimulated with IL-6 (50 ng/ml). Panel a represents a cell prior to IL-6 addition, and panel b represents the same cell 30 min after IL-6 addition. Boxed regions are magnified at right (panels a′ and b′). Panel c represents a kymograph from the region denoted with a white line. Scale bars, 10 μm. D, the number of filopodia was measured using live-cell imaging from eight cells from independent biological replicates using the FiloQuant program in ImageJ. The basal number of filopodia was set to 1 for each cell, and the graph represents the average relative number of filopodia following addition of IL-6. See also Fig. S1 and Movies S1 and S2.

Article Snippet: PANC-1 human pancreatic cancer cells were obtained from ATCC (CRL-1469).

Techniques: Migration, Transwell Migration Assay, Positive Control, Control, Incubation, Blocking Assay, Wound Healing Assay, Transfection, Fluorescence, Microscopy, Live Cell Imaging

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-6 promotes pancreatic cancer cell migration by rapidly activating the small GTPase CDC42

doi: 10.1074/jbc.RA118.003276

Figure Lengend Snippet: IL-6 stimulation leads to activation of CDC42 in pancreatic cancer cells. A, IL-6 rapidly activates CDC42. PANC-1 cells were treated with IL-6 for the indicated times (50 ng/ml), then lysed, and analyzed for CDC42 activation using a GST-PBD biochemical pulldown and Western blotting for CDC42. Note the rapid and robust activation of CDC42 within just 5 min post-stimulation. Active CDC42 was normalized to total CDC42 and compared with the ratio at t = 0. B, in contrast, IL-6 stimulation did not activate RAC1. PANC-1 cells were treated as described in A, and active RAC1 was precipitated using a GST-PBD pulldown followed by Western blotting for RAC1. Active RAC1 was normalized to total RAC1 and compared with the ratio at t = 0. C, IL-6 does not significantly activate RhoA. PANC-1 cells were stimulated with IL-6 as above, and active RhoA was precipitated using a GST-RBD pulldown followed by Western blotting for RhoA. Active RhoA was normalized to total RhoA and compared with the ratio at t = 0. Relative levels of RAC1, CDC42, or RhoA activation are graphed as the mean of three to six independent biological replicates. Error bars represent S.E. * indicates p < 0.05. See also Fig. S2.

Article Snippet: PANC-1 human pancreatic cancer cells were obtained from ATCC (CRL-1469).

Techniques: Activation Assay, Western Blot

Journal: Annals of Translational Medicine

Article Title: CMTM8 as an LPA1-associated partner mediates lysophosphatidic acid-induced pancreatic cancer metastasis

doi: 10.21037/atm-20-1013

Figure Lengend Snippet: Identification of CMTM8 as an LPA1-interacting protein. (A) PANC-1 and BxPC-3 cells were exposed to 10 μM lysophosphatidic acid (LPA) and immunoprecipitated with anti-LPA1 antibody or isotope control IgG and probed for indicated proteins by Western blotting. (B) Detection of LPA1 protein by Western blotting in CMTM8 immunoprecipitates from BxPC-3 and PANC-1 cells. (C) Confocal microscopy indicated that LPA1 and CMTM8 were co-localized in pancreatic cancer cells. Scale bar =10 μm. (D) Western blot analysis of LPA1 and CMTM8 in pancreatic cancer cell lines. All experiments were performed three times.

Article Snippet: Cell culture and treatment Human pancreatic cancer cell lines (BxPC-3, PANC-1, and CFPAC-1) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Immunoprecipitation, Control, Western Blot, Confocal Microscopy

Journal: Annals of Translational Medicine

Article Title: CMTM8 as an LPA1-associated partner mediates lysophosphatidic acid-induced pancreatic cancer metastasis

doi: 10.21037/atm-20-1013

Figure Lengend Snippet: CMTM8 augments the migratory and invasive capacity of pancreatic cancer cells. (A) Quantitative PCR analysis of CMTM8 mRNA levels in BxPC-3 and PANC-1 cells transfected with control or CMTM8 short hairpin RNAs (shRNAs). (B) In vitro wound-healing assay. The percentage of wound closure was evaluated 48 h after scratching. (C) Transwell invasion assay. After incubation for 48 h, invaded cells were stained with 0.1% crystal violet and counted using a microscope (×20). (D) Western blot analysis of CMTM8 protein levels in pancreatic cancer cells transfected with indicated constructs. Numbers indicate fold change. (E) In vitro wound-healing and (F) Transwell invasion assays were performed to assess the migration and invasion capacity, respectively, of CMTM8-overexpressing and control cells. *P<0.05. Data are expressed as mean ± SD (n=3).

Article Snippet: Cell culture and treatment Human pancreatic cancer cell lines (BxPC-3, PANC-1, and CFPAC-1) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Control, In Vitro, Wound Healing Assay, Transwell Invasion Assay, Incubation, Staining, Microscopy, Western Blot, Construct, Migration

Journal: Annals of Translational Medicine

Article Title: CMTM8 as an LPA1-associated partner mediates lysophosphatidic acid-induced pancreatic cancer metastasis

doi: 10.21037/atm-20-1013

Figure Lengend Snippet: CMTM8 knockdown inhibits pancreatic cancer metastasis in vivo. (A) CMTM8-depleted and control PANC-1 cells were inoculated to nude mice via the tail vein, and lung metastasis was analyzed by in vivo bioluminescence imaging. (B) Quantification of metastatic lesions (n=6 mice per group). (C) Representative hematoxylin and eosin stained sections of the lung with metastatic nodules. Scale bar =50 µm. (D) Determination of the numbers of metastatic nodules in the lung. *P<0.05. (E) Immunohistochemical analysis of CMTM8 in 64 pairs of pancreatic cancer and adjacent noncancerous pancreatic tissues. Right panels show semi-quantitative analysis of CMTM8 staining. Scale bar =100 µm.

Article Snippet: Cell culture and treatment Human pancreatic cancer cell lines (BxPC-3, PANC-1, and CFPAC-1) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, In Vivo, Control, Imaging, Staining, Immunohistochemical staining

Journal: Annals of Translational Medicine

Article Title: CMTM8 as an LPA1-associated partner mediates lysophosphatidic acid-induced pancreatic cancer metastasis

doi: 10.21037/atm-20-1013

Figure Lengend Snippet: CMTM8 is involved in lysophosphatidic acid (LPA)-mediated invasiveness in pancreatic cancer cells. (A,B) BxPC-3 and PANC-1 cells were transfected with control or CMTM8-targeting shRNA (shCMTM8#1) before exposure to LPA, and then subjected to (A) wound-healing and (B) Transwell invasion assays. *, P<0.05. (C) Western blot analysis of indicated protein levels. (D) BxPC-3 and PANC-1 cells were transfected with the TOP-FLASH or FOP-FLASH plasmid together with shCMTM8#1 before treatment with LPA. Luciferase activities were then measured. *, P<0.05. Data are expressed as mean ± SD (n=3).

Article Snippet: Cell culture and treatment Human pancreatic cancer cell lines (BxPC-3, PANC-1, and CFPAC-1) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection, Control, shRNA, Western Blot, Plasmid Preparation, Luciferase

Journal: Annals of Translational Medicine

Article Title: CMTM8 as an LPA1-associated partner mediates lysophosphatidic acid-induced pancreatic cancer metastasis

doi: 10.21037/atm-20-1013

Figure Lengend Snippet: CMTM8 promotes pancreatic cancer invasiveness by stimulating β-catenin activation. (A) Western blot analysis of indicated protein levels in pancreatic cancer cells transfected with CMTM8-expressing plasmid or empty vector. Numbers indicate fold change. (B) BxPC-3 and PANC-1 cells were transfected with the TOP-FLASH or FOP-FLASH plasmid together with CMTM8-expressing plasmid, and luciferase activities were measured. (C) Quantitative PCR analysis of β-catenin mRNA levels in BxPC-3 and PANC-1 cells transfected with control or β-catenin shRNAs. (D) In vitro wound-healing and (E) Transwell invasion assays were performed to assess the migration and invasion capacity, respectively, of pancreatic cancer cells transfected with indicated constructs. *P<0.05. Data are expressed as mean ± SD (n=3). (F) A schematic model showing the mechanism by which CMTM8 mediates lysophosphatidic acid (LPA)-induced invasiveness in pancreatic cancer cells.

Article Snippet: Cell culture and treatment Human pancreatic cancer cell lines (BxPC-3, PANC-1, and CFPAC-1) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activation Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Luciferase, Real-time Polymerase Chain Reaction, Control, In Vitro, Migration, Construct

Journal: International journal of oncology

Article Title: RRM1 is mediated by histone acetylation through gemcitabine resistance and contributes to invasiveness and ECM remodeling in pancreatic cancer.

doi: 10.3892/ijo.2023.5499

Figure Lengend Snippet: Figure 4. RRM1 gene silencing reduces cell migration and invasion. (A) Effects of RRM1 gene silencing on RRM1 expression. Cells were treated with RRM1‑specific siRNAs or negative control‑siRNA for 72 h. It was confirmed that RRM1 was suppressed by siRNA treatment in Panc1 and MIPaCa2 cells. (B) Effects of RRM1 gene silencing on cellular wound‑healing activity. Cancer cells were treated with siRRM1 or siNC for 24 h prior to wound scratch assay for 24 h. RRM1 gene silencing decreased the cellular migration of siRRM1 treated Panc1 cells compared with siNC control cells. Each assay was performed in duplicate. Occupancy of the wound area was also evaluated. The ability of migration was significantly decreased in siRRM1 cells. Error bars represent the mean ± SD. *P<0.05 by two‑way ANOVA. (C and D) Effects of RRM1 gene silencing on (C) cell motility and (D) invasion. Cells were treated with RRM1‑specific siRNAs or negative control‑siRNA for 24 h, then subjected to migration or invasion assays using Boyden chambers without or with Matrigel. Gene silencing of RRM1 reduced the cellular migration of two pancreatic cancer cell lines (46.9 and 43.7% decrease in Panc1 cells compared with siNC, 36.6 and 49.9% decrease in MIAPaCa2 cells, respectively; *P<0.05 by Tukey's test). Similarly, gene silencing of RRM1 reduced the cellular invasion of two pancreatic cancer cell lines (41.8 and 57.1% decrease in Panc1 cells, 36.6 and 49.8% in MIAPaCa2 cells, respectively; *P<0.05 by Tukey's post hoc test). Each assay was performed in duplicate. RRM1, ribonucleotide reductase large subunit M1; siRNA, small interfering RNA; NC, negative control.

Article Snippet: The human pancreatic cancer cell lines MIAPaCa2 and Panc1 were obtained from the American Type Culture Collection in August 2016.

Techniques: Migration, Expressing, Activity Assay, Wound Healing Assay, Control, Small Interfering RNA, Negative Control

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: PKM2 is a novel binding partner of PSAT1. ( A ) Silver stain of GST-PSAT1-purified proteins from A549 whole-cell lysates. * Denotes residual GST-PSAT1 from column purification; ← denotes gel slice encompassing PKM. ( B ) Primary amino acid sequence of human PKM. MS-identified peptides of PKM are highlighted in red. Black-labeled sequences belong to common regions of both PKM1 and PKM2 isoforms and green-labeled sequences identify isoform specificity. ( C ) Co-IP of recombinant (rec-) PSAT1 and PKM1 or PKM2. Immunocomplexes were precipitated using an anti-PSAT1 antibody and analyzed by immunoblot using anti-PKM1, anti-PKM2, and anti-PSAT1 antibodies. Recombinant proteins were used as input controls showing antibody specificity and PSAT1 alone was used as an IP control. Shown are representative images from two separate experiments.

Article Snippet: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Binding Assay, Silver Staining, Purification, Sequencing, Labeling, Co-Immunoprecipitation Assay, Recombinant, Western Blot

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Mutations within an isoform-specific region of PKM2 weakens the PSAT1 interaction. ( A ) Schematic representation of PKM2-specific mutations generated for the analysis of the PSAT1–PKM2 association. Ribbon representation of the structure of PKM2 colored by PKM1 sequence homology (right panel). Identical regions are shown in purple and divergent regions in cyan. The left panel depicts the site-directed mutagenesis of amino acids (MT1–4, highlighted in red) in the PKM2-specific region (denoted in the white box). ( B ) FLAG-PKM2 wild-type (WT), mutants (MT1–4) and FLAG-EV (-) were expressed in HEK293T cells. Endogenous PSAT1 protein complexes were immunoprecipitated and the association with PKM2 was assessed by immunoblot. A similar expression of FLAG-PKM2 variants is shown by immunoblot of FLAG fusion proteins from the protein lysate input with β-actin used for a protein loading control. Shown are representative images from three separate experiments. (-) denotes an empty vector.

Article Snippet: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Generated, Sequencing, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: PSAT1 associates with endogenous PKM2 in NSCLC cells but the loss of PSAT1 does not alter either PKM2 expression or pyruvate kinase activity. ( A ) Co-IP of PSAT1 and PKM2 in A549 and PC9 NSCLC cells. PSAT1-specific immunocomplexes were precipitated using anti-PSAT1 from a whole-cell lysate and analyzed for PKM2 by immunoblot with an anti-PKM2 antibody. Shown are representative images from three separate experiments. ( B ) Loss of PSAT1 expression in A549 and PC9 cells stably expressing PSAT1-specific shRNA. The PSAT1 expression was determined in whole-cell lysates from the control or shPSAT1-expressing cells by immunoblot using anti-PSAT1 and anti-α-tubulin (loading control). ( C ) Intracellular pyruvate kinase activity was determined in cell lysates from A549 or PC9 cells with or without PSAT1 expression. Data are represented as relative pyruvate kinase (PK) activity (control cells set to 1) and shown are the mean ± SD from four independent experiments. A statistical significance was determined by an unpaired t-test analysis. ( D ) Immunoblot analysis of PKM1 or PKM2 expression in whole-cell lysates from the control or PSAT1-silenced A549 and PC9 cells. Recombinant human PKM1 and PKM2 proteins were used as positive controls for antibody specificity and β-actin was used as a loading control. Shown are representative images from two separate experiments. IP: immunoprecipitation; IB: immunoblot; NS: not significant.

Article Snippet: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Activity Assay, Co-Immunoprecipitation Assay, Western Blot, Stable Transfection, shRNA, Recombinant, Immunoprecipitation

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Silencing of PSAT1 suppresses the nuclear localization of PKM2 in EGFR activated NSCLC cells. ( A ) EGFR-mutant PC9 cells stably expressing the control or PSAT1 shRNA were treated with 1 µM of erlotinib. Cytoplasmic and nuclear fractions were examined by an immunoblot analysis using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three separate experiments. ( B ) Nuclear localization of PKM2 was examined in serum-starved PC9 cells expressing the control or PSAT1 shRNA by confocal microscopy. DAPI served as a control for nuclear staining. Arrowheads indicate nuclear PKM2 staining in representative images from three independent experiments. ( C ) Serum-starved A549 cells (EGFR wild-type) stably expressing control or PSAT1 shRNA were treated with or without EGF (100 ng/mL). Cytoplasmic and nuclear fractions were prepared and the PKM2 and PSAT1 localizations were analyzed by immunoblot. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three separate experiments.

Article Snippet: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Mutagenesis, Stable Transfection, Expressing, shRNA, Western Blot, Confocal Microscopy, Staining

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Loss of PSAT1 decreases cell migration in EGFR-mutant PC9 and EGF-stimulated A549 NSCLC cells. ( A ) Wound healing assay of PC9 cells expressing the control or PSAT1-specific shRNA. Shown are representative images taken at 0 h and 24 h. Migrating cells are demarcated by continuous white lines. Data are presented as a migrated area after 24 h and shown is the mean ± SE from three independent experiments. A statistical significance was determined by a paired t-test analysis. * is p < 0.005. ( B ) Boyden chamber migration assay on serum-starved A549 cells expressing the control or PSAT1 shRNA. A total of 100 ng/mL EGF serum-free media was used as a chemo-attractant and migrated cells were fixed and stained with crystal violet after 24 h. Shown are representative images of the migrated cells and quantification is demonstrated as the mean ± SE of the% migration area from three independent experiments. A statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparison test. * p = 0.0001. N.S.: not significant; A.U.: arbitrary unit.

Article Snippet: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Migration, Mutagenesis, Wound Healing Assay, Expressing, shRNA, Staining

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Re-expression of PSAT1 restores the nuclear localization of PKM2 and cell migration in silenced PC9 cells. ( A ) Immunoblot analysis for PKM2 and PSAT1 localization in PSAT1-silenced PC9 cells stably expressing an empty vector (EV) or FLAG-PSAT1. Cytoplasmic and nuclear fractions from the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PSAT1 PC9 cells were analyzed using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three independent experiments. ( B ) Wound healing assay of the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PSAT1 PC9 cells. Shown are representative images at 0 h and 24 h. The migrating cells are demarcated by continuous white lines. Data are presented as a mean ± SE migrated area after 24 h from three independent experiments. A statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. ** p < 0.005 and * p < 0.05. A.U.: arbitrary unit.

Article Snippet: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Migration, Western Blot, Stable Transfection, Plasmid Preparation, Wound Healing Assay

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Re-expression of nuclear-localized acetyl-mimetic (K433Q) PKM2, but not wild-type PKM2, partially rescues the migration defect due to the loss of PSAT1. ( A ) Immunoblot analysis for PKM2 localization in PSAT1-suppressed PC9 cells stably expressing the nuclear-targeted wild-type PKM2 (FLAG-PKM2 NLS-WT ). Cytoplasmic and nuclear fractions from the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PKM2 NLS-WT -expressing cells were analyzed using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three independent experiments. ( B ) Wound healing assay of serum-starved PC9 cells expressing the control-EV, shPSAT1-EV, or shPSAT1-FLAG-PKM2 NLS-WT . Shown are representative images at 0 h and 24 h with migrating cells demarcated by continuous white lines. Data are presented as a mean ± SE migrated area after 24 h from three independent experiments. A statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. * p < 0.0001 and N.S.: not significant. ( C ) Immunoblot analysis for PKM2 localization in PSAT1-suppressed PC9 cells stably expressing nuclear-targeted acetyl-mimetic (K433Q) PKM2. Cytoplasmic and nuclear fractions from the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PKM2 NLS-K433Q -expressing cells were analyzed using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three independent experiments. ( D ) Wound healing assay of serum-starved PC9 cells expressing the control-EV or shPSAT1-EV and shPSAT1-FLAG-PKM2 NLS-K433Q . Shown are representative images at 0 h and 24 h with migrating cells demarcated by continuous white lines. Data are presented as a mean ± SE migrated area after 24 h from three independent experiments. A statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. ** p < 0.0001 and * p < 0.05. EV: empty vector; A.U.: arbitrary unit; NLS: nuclear localization signal.

Article Snippet: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Migration, Western Blot, Stable Transfection, Wound Healing Assay, Plasmid Preparation

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Schematic depicting putative nodes for PSAT1 regulation in the nuclear translocation and retention of PKM2 in response to EGFR activation. ( i ) PSAT1 interaction may promote cytoplasmic PKM2 phosphorylation and acetylation or suppress nuclear SIRT6-dependent deacetylation that contributes to nuclear PKM2 function in increasing pro-motility gene expression. ( ii ) PSAT1 may influence various steps involved in PKM2’s translocation and retention independent of direct PKM2 interaction. Dotted lines indicate putative PSAT1 regulatory steps.

Article Snippet: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Translocation Assay, Activation Assay, Expressing

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 1. TANGO1 expression in HCC samples A, B, C. TCGA database presented the increase of TANGO1 in HCC samples. D. Statistical results of TANGO1 expression and HIC score. E. Western blot detection TANGO1 expression in cell lines. F. Kaplan-Meier showing TANGO1 expression in TMA survival analysis. G. shRNA-TANGO1 and control lentivirus transfection HepG2 and Huh7. H. TANGO1 and vector lentivirus transfection HepG2 and Huh7. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: Expressing, Western Blot, shRNA, Control, Transfection, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 3. TANGO1 promotes HCC cell progression and inhibits apoptosis A, B. Cell cycle analysis of HepG2 and Huh7. C, D. Apoptosis levels of HepG2 and Huh7. E. Expression of cell cycle-related proteins verified by Western blot. *p < 0.05, **p < 0.01.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: Cell Cycle Assay, Expressing, Western Blot

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 5. TANGO1-interacting proteins and pathway enrichment A. RNA-seq of TANGO1 overexpression HepG2, volcano plot of DEGs obtained. B. GO functional enrichment to differential genes of BP, CC, MF. C. Biological pathways screened by KEGG functional analysis. D. qPCR validation of partial differential gene expression in TANGO1 overexpressing Huh7. E. TCGA database presented the increase of NRTN in HCC samples. F. Kaplan-Meier analysis of NRTN overall survival in HCC patients. *p < 0.05, **p < 0.01.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: RNA Sequencing, Over Expression, Functional Assay, Biomarker Discovery, Gene Expression

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 6. TANGO1 regulates the PI3K/AKT/mTOR pathway in HCC cells and interacts with NRTN A. Co-IP assay TANGO1/NRTN interaction in HepG2 and Huh7. B. Co-localization of TANGO1 and NRTN in HepG2 and Huh7. C, D. Western blot detect PI3K/AKT/mTOR pathway in HepG2 and Huh7. **p < 0.01, ***p < 0.001.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 7. NRTN silencing inhibits TANGO1-mediated proliferation and migration A. Western blot assay NRTN expression after siRNA transfection of HepG2 and Huh7. B. EdU assay showing silencing of NRTN inhibits HepG2 and Huh7 proliferation. C Wound healing assay showing silencing of NRTN inhibits HepG2 and Huh7 migration. D. Transwell assay showing silencing of NRTN inhibited HepG2 and Huh7 invasion. E. Western blot assay PI3K/AKT/mTOR marker expression in HepG2 and Huh7 after NRTN silencing. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: Migration, Western Blot, Expressing, Transfection, EdU Assay, Wound Healing Assay, Transwell Assay, Marker

Journal: Frontiers in Pharmacology

Article Title: USP5 Promotes Metastasis in Non-Small Cell Lung Cancer by Inducing Epithelial-Mesenchymal Transition via Wnt/β-Catenin Pathway

doi: 10.3389/fphar.2020.00668

Figure Lengend Snippet: High USP5 expression was correlated with EMT, invasion, and migration in NSCLC cells. (A) Western blot and (B) real-time PCR analysis of USP5 expression in NSCLC cell lines and normal lung fibroblast cells. GAPDH was used as an internal normalization control. The protein (C) and mRNA (D) levels of EMT markers were analyzed in NSCLC cells. (E) Migration of NSCLC cells was determined based on a wound-healing assay (top panel). Red bar, 200 μm. Statistical results were determined using an ANOVA (bottom panel). (F) Cell invasion was determined based on a transwell invasion assay (left panel) and assessed using an ANOVA (right panel). All results were obtained in five independent experiments (n = 5). NS = no statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001. USP5, ubiquitin-specific protease 5; EMT, epithelial–mesenchymal transition; NSCLC, non-small cell lung cancer; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ANOVA, analysis of variance.

Article Snippet: The normal human lung fibroblast WI-38 and NSCLC (W-358, A549, H1299, and 95-D) cell lines were purchased from the American Type Culture Collection and cultured under recommended conditions.

Techniques: Expressing, Migration, Western Blot, Real-time Polymerase Chain Reaction, Control, Wound Healing Assay, Transwell Invasion Assay, Ubiquitin Proteomics, Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: C -terminal amino acid Ala459 residue cooperates in the regulation of CAIX catalytic function: ( a ) Immunoblot analysis of cell lysates from C33a cells transfected with mock control, CAIX wild type (wt) or mutant (A459G) cultured 48 h in hypoxia. CAIX was detected using mouse monoclonal antibody M75 diluted 1:10 and β-actin was detected using mouse monoclonal antibody (CS3700, Cell Signaling Technology, Danvers, Massachusetts) diluted 1:5000. HRP-conjugated anti-mouse antibody (Dako Agilent, Santa Clara, California) diluted 1:5000 was used as a secondary antibody. ( b ) Fluorescence staining of CAIX in non-fixed and non-permeabilized C33a-CAIX wild type as well as A459G transfectants measured by flow cytometry. CAIX was detected using PG-domain specific mouse monoclonal antibody M75 diluted to 1 µg/mL and AlexaFluor 488-conjugated anti-mouse secondary antibody (Invitrogen, Carlsbad, California) diluted 1:1000. Results clearly demonstrate plasma membrane localization of CAIX and showed that 42.7% of C33a-CAIX-wt and 50.9% of C33a-CAIX-A459G transfectants expressed CAIX protein. C33a cells transfected with mock control plasmid were used as a negative control. The data are presented as the mean, n = 2. ( c ) Effect of Ala459 mutation on CAIX-mediated extracellular acidification. The graph shows the differences between pHe values (ΔpH) of culture media from CAIX wt or A459G-transfected and mock-transfected cells cultured 48 h in hypoxia. A459G mutant reduced acidification of extracellular pHe when compared to control wild type C33a-CAIX transfectants. The data are presented as the mean ± s.d., n = 5. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( d ) Effect of Ala459 mutation on migration capacity of C33a cells. The graph depicts the results of the wound healing assay given as a % of the area covered by cells migrating to close the wound at 30 h after the scratch, measured at various positions along the wounds. Area covered by C33a cells expressing CAIX-wt was set as 100%. C33a cells expressing CAIX with mutated Ala459 exhibited slower migration. The data are presented as the mean ± s.d., n = 10. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( e ) Accumulation of the fluorescent sulfonamide (FITC-CA-i) occurred in hypoxic MDCK cells expressing the CAIX-wt, whereas it was diminished in hypoxic MDCK cell transfected with the CAIX-A459G mutant. Images were taken using objective 10×. ( f ) In situ detection of the interaction between CAIX and AE2 using a proximity ligation assay (PLA). Analysis was performed in C33a cells transiently transfected with CAIX-wt and A459G mutant and cultured in hypoxia for an additional 48 h. Red PLA signal indicates the existence of interaction or close proximity localization also between CAIX with mutated Ala459 and AE2. C33a-mock transfectants served as a negative assay control due to a lack of one target protein. CAIX protein was post-labelled in green, nuclei are blue. Images were taken using objective 40× and zoom 3.

Article Snippet: Human C33a cervical carcinoma cells (ATCC HTB-31), HCT116 (ATCC CCL-247) and RKO (ATCC CRL-2577) colorectal carcinoma cells, MDA-MB-231 breast carcinoma cells (ATCC HTB-26), A549 lung carcinoma cells (ATCC CCL-185), Panc-1 (ATCC CLR-1469) pancreatic carcinoma and MDCK canine epithelial cells (ATCC CCL-34) were cultured under standard conditions in Dulbecco’s modified Eagle’s medium (BioSera, Nuaille, France) supplemented with 10% fetal calf serum (BioWhittaker, Basel, Switzerland) and gentamicine (Sandoz, Holzkirchen, Germany) in humidified air containing 21% O 2 , 5% CO 2 at 37 °C and in hypoxic conditions at anaerobic workstation (1% O 2 , 2% H 2 , 5% CO 2 , 91% N 2 , Ruskinn Technology, Bridgend, United Kingdom).

Techniques: Residue, Western Blot, Transfection, Control, Mutagenesis, Cell Culture, Fluorescence, Staining, Flow Cytometry, Clinical Proteomics, Membrane, Plasmid Preparation, Negative Control, Migration, Wound Healing Assay, Expressing, In Situ, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: WISP1 expression is increased in melanoma and is associated with reduced overall survival of patients diagnosed with primary melanoma. A, WISP1 mRNA expression in benign skin conditions (normal skin and benign melanocytic skin nevus) compared with primary melanoma. The original expression data set (GSE3189) was deposited by Talantov et al. (55). p values were calculated using analysis of variance with the post hoc Tukey honest significant difference 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 normal skin (left) and two melanoma (right) tissue samples. Original tissue microarray images were obtained from www.proteinatlas.org3 (77). Deconvoluted intensity of WISP1 staining is shown in red, whereas the cellular structures stained using hematoxylin are shown in blue. Arrows, melanocytes in epidermis; arrowheads, fibroblasts in dermis (stroma). C, average WISP1 staining within normal skin and primary melanoma tissue samples. D, distributions in nonzero pixel intensity values of WISP1 staining for normal skin (black curves) and primary melanoma (red curves) tissue samples. Numbers, percentage of the distribution that has pixel intensity values greater than a normalized pixel intensity of 0.2. E, Kaplan–Meier estimate of overall survival of melanoma patients stratified by WISP1 transcript abundance. The original data set was from the Cancer Genome Atlas. Sample numbers and p values calculated using the Peto and 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., p > 0.05).

Article Snippet: Two pairs of mouse Wisp1 double nickase plasmids (sc-423705-NIC and sc-423705-NIC-2), targeting the mouse Wisp1 gene at different locations, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX) and used in B16F10, YUMM1.7, and NIH3T3 cells.

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

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: WISP1 knockout in mouse and human melanoma cells inhibited tumor cell migration and invasion. A, 48-h 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 the left, and colony counts are 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 h. D, Boyden transwell migration assay of B16F10 and the two knockout cells. A representative staining image for each sample is shown on the left, and 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 concentrations 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 concentrations 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 p < 0.05 was considered significant, and asterisks were used to indicate calculated range in p values. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: Two pairs of mouse Wisp1 double nickase plasmids (sc-423705-NIC and sc-423705-NIC-2), targeting the mouse Wisp1 gene at different locations, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX) and used in B16F10, YUMM1.7, and NIH3T3 cells.

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

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

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 the indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (n = 5), and only mice surviving the whole experiments were analyzed at the same time for imaging, photography, and qPCR (final n ≥ 3). These experiments were repeated, and similar results were achieved. A, bioluminescence imaging performed 1 day before NSG mice were euthanized. All animals were compared with the same bioluminescence scale. B and 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 1 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). Four high-resolution images for B, D, F, and H are provided as Figs. S1–S4. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.D.

Article Snippet: Two pairs of mouse Wisp1 double nickase plasmids (sc-423705-NIC and sc-423705-NIC-2), targeting the mouse Wisp1 gene at different locations, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX) and used in B16F10, YUMM1.7, and NIH3T3 cells.

Techniques: Knock-Out, Injection, Imaging

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

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 the indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (n = 5), and two representative images are shown. A, bioluminescence imaging performed 1 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 was plotted and compared using bioluminescence intensity detected in A. Total flux is presented as photons/s (p/s). E, bioluminescence imaging performed 1 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, whole-body metastasis of tumor cells in C57BL/6Ncrl mice was plotted and compared using bioluminescence intensity detected in E. Two high-resolution images for B and F are provided as Figs. S7 and S8. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.D.

Article Snippet: Two pairs of mouse Wisp1 double nickase plasmids (sc-423705-NIC and sc-423705-NIC-2), targeting the mouse Wisp1 gene at different locations, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX) and used in B16F10, YUMM1.7, and NIH3T3 cells.

Techniques: Knock-Out, Injection, Imaging

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

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 h before they were harvested for RNA analysis or treated with the 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 a Boyden transwell invasion assay. B, immunoblot analysis of WISP1 protein to confirm the disruption of Wisp1 gene in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded in each lane, and β-actin was used as an internal loading control. B16F10-KO1-mWisp1 cells, in which mouse WISP1 expression was resumed with retroviral transduction, were used as a positive control. C, immunoblot analysis of certain EMT marker proteins in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded 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 concentration 5 μg/ml) and harvested at the 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 (CM). The conditioned media were pretreated with the indicated antibodies for 30 min before they were used on Wisp1-knockout B16F10 cells (-KO1). The cells were collected for real-time qRT-PCR after 3 h of treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: Two pairs of mouse Wisp1 double nickase plasmids (sc-423705-NIC and sc-423705-NIC-2), targeting the mouse Wisp1 gene at different locations, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX) and used in B16F10, YUMM1.7, and NIH3T3 cells.

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

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: SNAI1 overexpression in B16F10 Wisp1-knockout cell rescued the repression of 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 h before they were 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 the left, and relative invasion efficiency is graphed on the right. D, experimental metastasis assay in NSG mice using the indicated cells. Each group contained 3–4 mice. All mice were imaged 1 day before the end of the assay, and representative bioluminescence images are shown. E, representative lung and liver images from NSG mice in the experimental metastasis assay described in D. Metastatic tumor colonies on the lung surface from mice with -mWisp1 or -hSnai1 cells are indicated by arrows. F, real-time genomic qPCR for lungs and livers from the experimental metastasis assay in D. The quantitative tumor metastatic burdens were presented as tumor cell number within 10,000 mouse tissue cells. A high-resolution image for E is provided as Fig. S9. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: Two pairs of mouse Wisp1 double nickase plasmids (sc-423705-NIC and sc-423705-NIC-2), targeting the mouse Wisp1 gene at different locations, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX) and used in B16F10, YUMM1.7, and NIH3T3 cells.

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

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: WISP1 activated AKT and MEK/ERK signaling and promoted EMT marker gene expression in mouse melanoma cells. Unless otherwise specified, cell treatment for kinase immunoblot analysis was maintained for 30 min before cells were lysed for protein extraction, whereas cell treatment for comparison of EMT marker gene expression was maintained for 3 h before cells were harvested for RNA extraction. A, comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in B16F10 cells. DMSO was used for control cells. Immunoblotting for phospho-AKT (pAKT) and phospho-ERK1/2 (pERK1/2) is shown in the top right corner. Pan-AKT and total ERK1/2 were probed as loading control. B, comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in YUMM1.7 cells. C, immunoblot analysis of AKT and ERK1/2 activation in the indicated mouse melanoma cells with treatment of recombinant mouse WISP1 protein (rmWISP1; final concentration 5 μg/ml). All cells were grown on 6-well plates in complete DMEM for 48 h and SFM for another 48 h before rmWISP1 was added. D, immunoblot analysis of AKT and ERK1/2 activation in B16F10 knockout cell (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were grown on 6-well plates in complete DMEM for 48 h (0-h point for SFM) and switched to SFM for 24 or 48 h. The indicated cells were treated with rmWISP1 at the 0-, 24-, and 48-h time points (of SFM) for 30 min before they were lysed for kinase analysis. The first lane on the gels was loaded with YUMM1.7 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. E, immunoblot analysis of AKT and ERK1/2 activation in YUMM1.7 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D. The first lane on the gels was loaded with B16F10 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. F, comparison of SNAI11 activation and E-cadherin repression in B16F10 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D except that rmWISP1 treatment at each point was maintained for 3 h. G and H, comparison of EMT marker gene expression after AKT/ERK1/2 activation in B16F10-KO1 (G) or YUMM1.7-KO1 (H) by rmWISP1 was blocked. rmWISP1 with DMSO or inhibitors was added after the indicated cells were grown on 6-well plates in complete DMEM for 48 h and in SFM for 24 h. The relative protein levels of pAKT and AKT and of pERK1/2 and ERK1/2 in C–E were measured, and they are listed in Table S3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: Two pairs of mouse Wisp1 double nickase plasmids (sc-423705-NIC and sc-423705-NIC-2), targeting the mouse Wisp1 gene at different locations, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX) and used in B16F10, YUMM1.7, and NIH3T3 cells.

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