Journal: iScience

Article Title: The Taspase1/Myosin1f-axis regulates filopodia dynamics

doi: 10.1016/j.isci.2022.104355

Figure Lengend Snippet: Myo1f is a bona fide substrate of Tasp1 (A/B) A semi-in vitro Tasp1 substrate cleavage assay was employed to verify the predicted Tasp1 cleavage site of Myo1f. 293T cells were transfected with plasmids encoding Myo1f-GFP or the cleavage site-deficient mutant Myo1f- 245 AA 246 -GFP. 24 h after transfection, cell lysates were incubated with or without 10 μM recombinant Tasp1-His at 37°C for the indicated time periods. The assay was performed without (A) or with (B) a two-fold concentrated protease inhibitor mix. Immunoblot analysis revealed the appearance of the C-terminal cleavage product (calculated molecular weight: 124 kDa) only in case of wild-type Myo1f and Tasp1-His presence confirming a Tasp1-His mediated cleavage of full-length (FL) Myo1f-GFP. (C) Schematic tripartite structure of Myo1f comprising a head (blue oval), a neck (blue sphere) and a tail (gray) domain. Indicated are the proposed nuclear localization signal (NLS) and the Tasp1 cleavage site (CS), the ATP and actin binding domains located within the head domain, and the nuclear export signal (NES)-bearing region (red) arranged between the IQ calmodulin-binding motif, the motor and tail homology 1 and 2 (TH1/TH2) and src homology 3 domains as part of the tail. (D) 293T cells co-expressing the indicated Myo1f variants and Tasp1-HA were treated with 50 nM bortezomib. Immunoblotting of whole cell extracts revealed a lower migrating band only for WT Myo1f-GFP after proteasome inhibition.

Article Snippet: H.sapiens: HEK 293T , ATCC , CRL-3216; RRID: CVCL_0063.

Techniques: In Vitro, Cleavage Assay, Transfection, Mutagenesis, Incubation, Recombinant, Protease Inhibitor, Western Blot, Molecular Weight, Binding Assay, Expressing, Inhibition

Journal: iScience

Article Title: The Taspase1/Myosin1f-axis regulates filopodia dynamics

doi: 10.1016/j.isci.2022.104355

Figure Lengend Snippet: Myo1f is a nucleo-cytoplasmic shuttle protein (A) Schematic presentation of full-length Myo1f-GFP (left) and myc-/GFP-labeled Myo1f-truncations representing the N- (middle) and the C-terminal Tasp1 cleavage product (right). (B) Confocal fluorescence microscopy images showing the subcellular localization of Myo1f and truncated variants in HeLa cells. Full length Myo1f-GFP exhibited a predominantly cytosolic localization, but exposure to Leptomycin B (LMB) results in a shift to an increased nuclear localization (left panel). In contrast, the N-terminal fragment clearly localized to the nucleus, whereas the C-terminal fragment was exclusively cytoplasmic (right panel). Scale bars 25 μm. (C) Quantification of the intracellular localization of full-length Myo1f-GFP using the software “CellProfiler3” ( Schindelin et al., 2012 ). Mean nuclear fluorescence intensity between 500 nm and 550 nm was measured in ROIs defined by Hoechst staining. Error bars represent the CI 95%, asterisks indicate statistical significance of mean difference assessed by t-test (∗: p ≤ 0.05; ∗∗: p ≤ 0.01; ∗∗∗: p ≤ 0.001). (D) Gaussian fit of the frequency distributions of measured mean nuclear fluorescence intensities further substantiated the increased nuclear localization upon LMB treatment, as two distinct populations become apparent. n = 2,572 cells from 4 independent experiments. (E) Myo1f-GFP and Myo1f- 245 AA 246 -GFP interact with RNA polymerase II and Histone H3. Co-immunoprecipitation of GFP-tagged Myo1f-variants from 293T chromatin fractions as detected by immunoblot analysis. Input = chromatin fraction; eluate, 40× concentrated. See also Figures S1 and .

Article Snippet: H.sapiens: HEK 293T , ATCC , CRL-3216; RRID: CVCL_0063.

Techniques: Labeling, Fluorescence, Microscopy, Software, Staining, Immunoprecipitation, Western Blot

Journal: iScience

Article Title: The Taspase1/Myosin1f-axis regulates filopodia dynamics

doi: 10.1016/j.isci.2022.104355

Figure Lengend Snippet: Myo1f-GFP induces filopodia-like membrane protrusions (A) Myo1f-GFP expressing HeLa cells are characterized by increased filopodia formation as detected by spinning disc confocal microscopy. Scale bars 25 μm. (B) FiloQuant analysis of relative filopodia density (filopodia number per μm cell surface) in HeLa cells expressing Myo1f-GFP compared to GFP expressing control cells. Statistical significance was assessed by t-test with a total of n = 100 cells/group in 3 independent experiments (∗: p < 0,05). Error bars represent the standard deviation. (C) FiloQuant measurement of filopodia longer than 1 μm in HeLa cells expressing Myo1f-GFP compared to GFP expressing control cells. n = 100 cells/group from 3 independent experiments. (D) Formation of Myo1f-GFP-induced cell protrusions containing filamentous F-actin in different adherent cell lines. 293T-, HeLa- and SW480 cells were transfected with either Myo1f-GFP (left panel) or GFP (right panel) and fixed 24 h later. F-actin was stained with rhodamine-conjugated phalloidin (red) and cells were analyzed by laser scanning confocal microscopy. Scale bars 25 μm. (E) THP-1 monocytes (Mo) were differentiated into macrophage-like cells (THP-1 macrophages, Mφ) by PMA treatment and analyzed by immunostaining. Endogenous Myo1f of THP-1 macrophages localizes to cell protrusions together with filamentous actin whereas THP-1 monocytes show comparatively less endogenous Myo1f and no filopodia. Scale bars 25 µm. (F) Immunoblot showing varying Tasp1 expression in different cell lines. (G) Decreased Tasp1 expression coincides with increased full-length Myo1f protein levels during differentiation from monocytes (Mo) into macrophages (Mφ) as demonstrated by immunoblot analysis. See also Figures S3 and .

Article Snippet: H.sapiens: HEK 293T , ATCC , CRL-3216; RRID: CVCL_0063.

Techniques: Membrane, Expressing, Confocal Microscopy, Control, Standard Deviation, Transfection, Staining, Immunostaining, Western Blot

Journal: iScience

Article Title: The Taspase1/Myosin1f-axis regulates filopodia dynamics

doi: 10.1016/j.isci.2022.104355

Figure Lengend Snippet: Tasp1 co-expression reduces filopodia in cells expressing Myo1f but not in cells expressing a non-cleavable Myo1f mutant (A/B) Counting of filopodia longer than 5 μm in HeLa (A) and 293T (B) cells expressing Myo1f- or Myo1f- 245 AA 246 -GFP together with either Tasp1-mCherrry (n = 83/n = 87) or a mock plasmid (n = 114/n = 94); filopodia counts per cell were normalized to the respective control cells without Tasp1 co-expression. Significance was analyzed by an unpaired t-test (∗∗: p ≤ 0.01; ns: not significant). Error bars indicate the standard deviation. (C) Representative confocal microscopy images of cells expressing the indicated constructs. Scale bars 25 μm.

Article Snippet: H.sapiens: HEK 293T , ATCC , CRL-3216; RRID: CVCL_0063.

Techniques: Expressing, Mutagenesis, Plasmid Preparation, Control, Standard Deviation, Confocal Microscopy, Construct

Journal: iScience

Article Title: The Taspase1/Myosin1f-axis regulates filopodia dynamics

doi: 10.1016/j.isci.2022.104355

Figure Lengend Snippet: Myo1f facilitates cellular adhesion and migration (A) Myo1f-induced filopodia actively sense their environment. Representative TIRF images of cells overexpressing Myo1f-GFP or GFP control. Yellow box indicates zoomed time-lapse sequential TIRF images which show a dynamic alternation of filopodia growth and retraction as indicated by the white arrows. Scale bars 25 μm. (B) Overexpression of Myo1f-GFP enhances the adhesion of 293T cells on fibronectin-coated plates as assessed by a MTS assay at the indicated time points. This effect was significantly counteracted by co-expression of Tasp1-mCherry. Error bars show the SD (n = 3), asterisks indicate significant differences in mean comparison by two-Way ANOVA(∗: p ≤ 0.05; ∗∗∗∗: p ≤ 0.0001). (C) Overexpression of Myo1f-GFP significantly increases the migration capacity of 293T cells as assessed by a Boyden chamber assay. Error bars show the SD (n = 3), asterisks indicate significant differences evaluated by two-Way ANOVA (∗∗: p ≤ 0.01, ∗∗∗∗: p ≤ 0.001).

Article Snippet: H.sapiens: HEK 293T , ATCC , CRL-3216; RRID: CVCL_0063.

Techniques: Migration, Control, Over Expression, MTS Assay, Expressing, Comparison, Boyden Chamber Assay

Journal: iScience

Article Title: The Taspase1/Myosin1f-axis regulates filopodia dynamics

doi: 10.1016/j.isci.2022.104355

Figure Lengend Snippet:

Article Snippet: H.sapiens: HEK 293T , ATCC , CRL-3216; RRID: CVCL_0063.

Techniques: Recombinant, Virus, Western Blot, Migration, Extraction, Isolation, Amplification, Plasmid Preparation, Software

Journal: Journal of Cancer

Article Title: OCT4-mediated upregulation of DUSP6 promotes metastasis in non-small-cell lung cancer

doi: 10.7150/jca.108663

Figure Lengend Snippet: Expression of OCT4 and DUSP6 is positively correlated in lung adenocarcinoma (LUAD) cells. (A) Immunohistochemical (IHC) staining and quantification of paraffin-embedded tumor parts and normal tissue sections for OCT4 and DUSP6 expression (× 200 magnification, scale bar = 50 μm). IHC intensity was quantified using the ImageJ software. Values shown are levels of immunointensity in individual specimens, with the mean level in normal tissue arbitrarily set to 100, in three randomly selected fields in each section (n=3). (B) A positive correlation between the expression of POU5F1 (OCT4) and DUSP6 in the expression profiles of 226 pathological stage I-II lung adenocarcinomas (NCBI Gene Expression Omnibus: GSE31210 ). The correlation was assessed using Pearson's correlation coefficient ( r =0.1268, P =0.0439). (C) Immunoblot analysis and quantification of endogenous expression levels of OCT4 and DUSP6 in eight lung cancer cell lines. Expression of β-actin served as the loading control. (D) Positive correlation between OCT4 and DUSP6 expression levels in cancer cell lines. Band intensity was quantified using the ImageJ software, and correlations were assessed using Pearson's correlation coefficient ( r= 0.8265 , P= 0.0017). (E, F) Increased expression of DUSP6 mRNA (E) and protein (F) in lung cancer cells overexpressing OCT4 as examined by RT-PCR (E, left ), quantitative real-time PCR (qPCR) (E, right ), and immunoblotting (F). A549 and H1299 cells were transduced with lentiviral vectors encoding OCT4 or luciferase, followed by puromycin selection. Expression of GAPDH (E) and β-actin (F) served as the internal control. (G, H) Knockdown of OCT4 by OCT4 shRNA reduces DUSP6 expression. A549 cells were transduced with lentiviral vectors expressing shRNAs specific to OCT4 (shOCT4 #2 and shOCT4 #5) or to luciferase (shLuc). Levels of OCT4 and DUSP6 mRNA and protein were examined by RT-PCR (G, left ), qPCR (G, right ), and immunoblotting (H). (I, J) Enhanced migratory and invasive capability of OCT4-overexpressing A549 cells, or reduced that of OCT4-knockdown A549 cells. Migration of A549/OCT4 and A549/Vector cells or A549/shLuc and A549/shOCT4#5 cells was detected by the Boyden chamber assay. Cells that migrated through the membrane of the lower surface in the Boyden chamber were stained with Giemsa solution and visualized by light microscopy (× 200 magnification, scale bar = 200 μm) (I, J, left ) and quantified (I, J, right ). Migratory cells in three fields in each membrane were counted. All quantification values and error bars of (A) and (E-J) shown were mean ± SEM. SEM, standard error of the mean.

Article Snippet: Human lung cancer cell lines A549 (ATCC CCL-185), H1299 (ATCC-CRL-5803), and H661 (ATCC-HTB-183TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Software, Gene Expression, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transduction, Luciferase, Selection, Knockdown, shRNA, Migration, Plasmid Preparation, Boyden Chamber Assay, Membrane, Staining, Light Microscopy

Journal: Journal of Cancer

Article Title: OCT4-mediated upregulation of DUSP6 promotes metastasis in non-small-cell lung cancer

doi: 10.7150/jca.108663

Figure Lengend Snippet: OCT4 upregulates DUSP6 expression by transactivating the DUSP6 promoter in lung cancer cells. (A) Reporter assay of the DUSP6 promoter in lung cancer cell lines. A549, H1299, and CL1-5 cells were co-transfected with pFRL2-hDUSP6p-Luc and pCMV-tag2B-hOCT4 (or pCMV-tag2B). (B) Identification of OCT4 binding sites on the human DUSP6 promoter. Schematic representation of a series of deletion constructs containing different lengths of the DUSP6 promoter. H1299 cells were co-transfected with different deletion reporter constructs of pFRL2-hDUSP6p-Luc and pCMV-tag2B-hOCT4 (or pCMV-tag2B). (C) A549 cells were co-transfected with pFRL2-hDUSP6p-Luc carrying a point mutation within the -423 ~ -416 ORE and pCMV-tag2B-hOCT4 (or pCMV-tag2B). Numbering is relative to the translational start site at +1. The white box indicates putative OREs. Cell lysates were harvested 48 h after transfection, and their firefly and Renilla luciferase activities were determined using a dual-light luciferase reporter assay system. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light units (RLU). Data are expressed relative to the activity of the full-length promoter construct (B) or the wild-type (WT) promoter construct (C) obtained from three independent experiments (B). (D) ChIP assay showing direct binding of OCT4 to the ORE located at -423 to -416 within the human DUSP6 promoter. Cross-linked chromatin of H1299 cells was immunoprecipitated with mouse anti-human OCT4 antibody or normal mouse IgG in combination with protein G agarose beads, followed by PCR amplification of the DUSP6 promoter region encompassing OCT4 binding sites.

Article Snippet: Human lung cancer cell lines A549 (ATCC CCL-185), H1299 (ATCC-CRL-5803), and H661 (ATCC-HTB-183TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Reporter Assay, Transfection, Binding Assay, Construct, Mutagenesis, Luciferase, Activity Assay, Immunoprecipitation, Amplification

Journal: Journal of Cancer

Article Title: OCT4-mediated upregulation of DUSP6 promotes metastasis in non-small-cell lung cancer

doi: 10.7150/jca.108663

Figure Lengend Snippet: Overexpression of OCT4 in A549 cells promotes tumor growth and metastasis and increases DUSP6 expression in mice. (A) Tumor volumes of mice bearing OCT4-overexpressing A549 tumors. NOD/SCID mice were subcutaneously inoculated with 1 × 10 6 cells of A549 cells overexpressing OCT4 (n=4 for OCT4 #4 and OCT4 #5) or vector control (n=4) A549 cells on day 0. Tumor volumes of the mice were measured. Tumor volumes were plotted through day 120 (primary tumor endpoint), and lungs were collected at day 156 to permit the development of spontaneous metastases in this slow‑metastasizing A549 model. (B) Number of metastatic nodules in the lung of individual mice. (C) Representative gross and histologic (hematoxylin and eosin stain, × 100 magnification, scale bar = 1.0 mm) appearances of lungs on day 156. Arrows denote tumor nodules. (D) Immunohistochemical detection of OCT4 and DUSP6 in paraffin-embedded lung tissue sections (× 100 magnification, scale bar = 100 μm).

Article Snippet: Human lung cancer cell lines A549 (ATCC CCL-185), H1299 (ATCC-CRL-5803), and H661 (ATCC-HTB-183TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, H&E Stain, Immunohistochemical staining

Journal: Journal of Cancer

Article Title: OCT4-mediated upregulation of DUSP6 promotes metastasis in non-small-cell lung cancer

doi: 10.7150/jca.108663

Figure Lengend Snippet: The OCT4-DUSP6 axis promotes the migratory and invasive capability of A549 cells. (A) OCT4-DUSP6 axis upregulates the expression of MMP2 and MMP9. A549 cells were transduced with lentiviral vectors encoding OCT4 or GFP (control), followed by puromycin selection. Subsequently, the cells were further transduced with lentiviral vectors encoding shRNA specific to DUSP6 (shDUSP6 #2 and shDUSP6 #4) or luciferase (shLuc). Detection and quantification of OCT4, DUSP6, MMP2, and MMP9 protein expression levels were examined by immunoblotting (n=3). Expression of β-actin served as the loading control. (B) The OCT4-DUSP6 axis regulates MMP2 and MMP9 expression independently of the inhibition of ERK1/2-MAPK activity. OCT4 or GFP-transduced A549 cells were treated with DMSO or ERK inhibitor (LY3214996, 2μM) for 48 h. Total protein extracts were detected and quantified for OCT4, DUSP6, NICD, MMP2, and MMP9 expression (n=3). (C) The OCT4-DUSP6 axis regulates MMP2 and MMP9 expression through the Notch pathway. A549 cells were transduced with lentiviral vectors encoding OCT4 or GFP, followed by further transduction with lentiviral vectors encoding shRNA specific to NOTCH (shNOTCH) or luciferase (shLuc). Detection and quantification of OCT4, DUSP6, NICD, MMP2, and MMP9 protein expression levels were examined by immunoblotting (n=3). (D) Migratory and invasive capabilities of DUSP6-knockdown and Luc control A549/OCT4 cells. Cells that migrated through the membrane to the lower surface in the Boyden chamber were stained with Giemsa solution and visualized by light microscopy (× 200 magnification, scale bar = 200 μm) (B) and quantified (C). Migratory cells in three fields in each membrane were counted. All quantification values and error bars shown were mean ± SEM, and P-values less than 0.05 were shown in the quantitative charts. NICD, Notch intracellular domain; SEM, standard error of the mean.

Article Snippet: Human lung cancer cell lines A549 (ATCC CCL-185), H1299 (ATCC-CRL-5803), and H661 (ATCC-HTB-183TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Transduction, Control, Selection, shRNA, Luciferase, Western Blot, Inhibition, Activity Assay, Knockdown, Membrane, Staining, Light Microscopy

Journal: Journal of Cancer

Article Title: OCT4-mediated upregulation of DUSP6 promotes metastasis in non-small-cell lung cancer

doi: 10.7150/jca.108663

Figure Lengend Snippet: Knockdown of DUSP6 in OCT4-overexpressing A549 cells decreases tumor growth and metastasis in mice. (A) Tumor volumes of mice bearing DUSP6-knockdown or vector control A549/OCT4 tumors. NOD/SCID mice were subcutaneously inoculated with 1 × 10 6 cells of DUSP6-knockdown (n=6 for OCT4_shDUSP6#2; n=7 for OCT4_shDUSP6#4) or vector control A549/OCT4 cells (n=7 for OCT4_shLuc). Tumor volumes of the mice were measured. (B) Number of metastatic nodules in the lung of individual mice. (C) Representative gross and histologic (hematoxylin and eosin stain, × 200 magnification, scale bar = 1.0 mm) appearances of lungs on day 86. Arrows denote tumor nodules. ( D ) A schematic representation of the OCT4-DUSP6 axis involved in lung cancer progression. OCT4 overexpression in NSCLC cells upregulates DUSP6. DUSP6 has been shown to function as a phosphatase for Notch1, thereby regulating Notch1 transmembrane/intracellular region (NTM) stability and Notch1 intracellular domain (NICD) transcriptional activity, resulting in driving tumor aggressiveness through MMP2 and MMP9 upregulation. The pathways in gray color have been reported previously.

Article Snippet: Human lung cancer cell lines A549 (ATCC CCL-185), H1299 (ATCC-CRL-5803), and H661 (ATCC-HTB-183TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Control, H&E Stain, Over Expression, Activity Assay

Journal: International Journal of Oncology

Article Title: Galectin-1 is a diagnostic marker involved in thyroid cancer progression

doi: 10.3892/ijo.2017.4065

Figure Lengend Snippet: Impact of galectin-1 depletion on 8505C cell proliferation and invasion. (A) Effect of galectin-1 knockdown on 8505C cell growth determined by cell counting during 7 days. Data are mean values of triplicates ± SD. (B and C) Effect of galectin-1 depletion on the invasion capacities of 8505C cells determined by Boyden chamber assay. Inserts of Boyden chambers were seeded with scrambled (gal-1 sc) and knockdown (gal-1 KD) cells (four inserts per group). Invasive cells on the lower membrane surface were determined by cell counting after crystal violet staining (Magnification, ×100). Data are presented as individual values (quadruplicate for both 8505C gal-1 sc and 8505C gal-1 KD). (D) Detection of matrix metallopeptidase 9 (MMP9) expression by immunofluorescence in the cytoplasmic compartment of 8505C gal-1 sc and 8505C gal-1 KD cells; cell nuclei are stained by DAPI (blue staining). Magnification ×400.

Article Snippet: The following primary antibodies were used: galectin-1 (polyclonal rabbit anti-human galectin-1 at 1:100, 1 h) ( , ) and matrix metalloproteinase 9 (MMP9) (polyclonal rabbit anti-MMP9 at 1:50, 1 h; sc-10737, Santa Cruz Biotechnology).

Techniques: Knockdown, Cell Counting, Boyden Chamber Assay, Membrane, Staining, Expressing, Immunofluorescence