Journal: NPJ Biofilms and Microbiomes

Article Title: Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium

doi: 10.1038/s41522-022-00290-3

Figure Lengend Snippet: NHNE cells from five healthy volunteers were inoculated with human nasal S. epidermidis at an MOI of 0.25. A FemA mRNA levels, normalized to cellular GAPDH transcript levels, were monitored by real-time PCR. B S. epidermidis CFUs were determined at 1 dpi in the supernatant of S. epidermidis -inoculated NHNE cells. C Dot plot visualization of enriched GO terms in S. epidermidis– inoculated NHNE cells at 1 dpi. D Scatterplots indicating enriched genes correlated with serine-type peptidase inhibitors. E Expressions of Serpine1 and Serpine2 from scRNA-seq of NHNE cells. Dot size represents the proportion of Serpines RNA expression within the respective cell type expressing the gene, and dot color represent the average Serpines RNA expression level in the particular cell type. F Serpine1 and Serpine2 mRNA levels, normalized to cellular GAPDH transcript levels, were monitored by real-time PCR (blue bar: Serpine1; red bar: Serpine2). G The intracellular protein level of Serpine1 was measured in the cell lysate of S. epidermidis– inoculated NHNE cells using western blot analysis. H The secreted protein level of Serpine1 was measured in the supernatant of S. epidermidis -inoculated NHNE cells using ELISA. I Heatmap depicting the expression levels of genes related with serine-type peptidase inhibitor activity differentially expressed in S. epidermidis -inoculated NHNE cells depending on the cellular subset. J Serpine1 mRNA level, normalized to cellular GAPDH transcript levels, was monitored by real-time PCR over 1 day after IAV infection following S. epidermidis inoculation. K , L The neutralizing antibody for Serpine1 was administered to NHNE cells one hour before S. epidermidis inoculation, and then the cells were infected with IAV for 1 day. IAV mRNA and NPs of IAV were compared in IAV-infected NHNE cells after S. epidermidis inoculation depending upon the neutralization of Serpine1. Results are presented as mean ± SD values from five independent experiments. * p < 0.05 vs. mock-infected NHNE cells.

Article Snippet: Primers for human or mouse Serpine1 (assay ID Hs00167155_m1, Mm00435858_m1), Serpine2 (assay ID Hs00299953_m1, Mm00436753_m1), TMPRSS11E (assay ID Hs01070171_m1, Mm01212186_m1), TMPRSS11F (assay ID Hs01592083_m1, Mm00812591_m1), TMPRSS2 (assay ID Hs01122322_m1, Mm00443687_m1), and KLK5 (assay ID Hs01548153_m1, Mm01203811_m1) were purchased from Applied Biosystems (Foster City, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Infection, Neutralization

Journal: NPJ Biofilms and Microbiomes

Article Title: Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium

doi: 10.1038/s41522-022-00290-3

Figure Lengend Snippet: Differential expression gene (DEG) linked with serine-type peptidase inhibitors in S. epidermidis -inoculated NHNE cells.

Article Snippet: Primers for human or mouse Serpine1 (assay ID Hs00167155_m1, Mm00435858_m1), Serpine2 (assay ID Hs00299953_m1, Mm00436753_m1), TMPRSS11E (assay ID Hs01070171_m1, Mm01212186_m1), TMPRSS11F (assay ID Hs01592083_m1, Mm00812591_m1), TMPRSS2 (assay ID Hs01122322_m1, Mm00443687_m1), and KLK5 (assay ID Hs01548153_m1, Mm01203811_m1) were purchased from Applied Biosystems (Foster City, CA, USA).

Techniques: Quantitative Proteomics

Journal: NPJ Biofilms and Microbiomes

Article Title: Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium

doi: 10.1038/s41522-022-00290-3

Figure Lengend Snippet: A Scatterplots indicating enriched genes related with serine-type peptidase. B PPI network of DEGs linked with serine-type peptidase indicates the protein – protein interaction with Serpine1 and Serpine2 (PP protein – protein). C Heatmap depicting the expression levels of genes related with serine-type peptidase inhibitor activity differentially expressed in S. epidermidis– inoculated NHNE cells depending on a cellular subset. D Serpine1 mRNA and E protein levels were monitored by real-time PCR and western blot analysis following S. epidermidis inoculation at 1 dpi with transfection with cont shRNA and uPA shRNA. F Serpine1 (blue bar) and PLAU mRNAs (red bar) were compared in NHNE cells after S. epidermidis inoculation. G ELISA results showed the secreted protein levels of Serpine1 (blue bar) and uPA (red bar) in S. epidermidis -inoculated NHNE cells. H The intracellular protein levels of HAT and Serpine1 were measured in the cell lysate of S. epidermidis– inoculated NHNE cells at 4, 8, and 24 h using western blot analysis. I The mRNA levels of proteases, such as TMPRSS2, TMPRSS11E, TMPRSS11F, and KLK5, which were targeted by Serpine1, were measured using the cell lysate of S. epidermidis -inoculated NHNE cells at 4, 8, and 24 h. Western blot results are representative of five independent experiments. Real-time PCR and plaque assay results are presented as mean ± SD values from five independent experiments. * p < 0.05 vs. mock-infected NHNE cells.

Article Snippet: Primers for human or mouse Serpine1 (assay ID Hs00167155_m1, Mm00435858_m1), Serpine2 (assay ID Hs00299953_m1, Mm00436753_m1), TMPRSS11E (assay ID Hs01070171_m1, Mm01212186_m1), TMPRSS11F (assay ID Hs01592083_m1, Mm00812591_m1), TMPRSS2 (assay ID Hs01122322_m1, Mm00443687_m1), and KLK5 (assay ID Hs01548153_m1, Mm01203811_m1) were purchased from Applied Biosystems (Foster City, CA, USA).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Transfection, shRNA, Enzyme-linked Immunosorbent Assay, Plaque Assay, Infection

Journal: Oncotarget

Article Title: Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

doi: 10.18632/oncotarget.12927

Figure Lengend Snippet: ( A ) Masson trichrome staining performed on 4T1 Ctrl and shSerpine2 tumors. Representative images show increased collagen encapsulation (blue) of SerpinE2 KD tumors. Scale bar100μm. ( B - C ) Intravital imaging using (IVI-MP) of 4T1 Ctrl and shSerpine2 tumors. (B) Representative images show collagen I fibers detected by the SHG signal (cyan) at the surface of 4T1 tumors; scale bar25μm. (C) Average SHG signal intensity was determined per 100μm z-stack; data are acquired from 19-30 separate z-stacks from 3 mice per cell line. * P < 0.00036. D. IVI-MP performed on mice bearing GFP-labeled 4T1 control and shSerpinE2 tumors. Representative images show GFP-labeled tumor cells (green), phagocytic dextran positive cells (red); SHG imaging identified collagen I fibers (cyan). Scale bars25μm. (E-F) ( E ) GFP-labeled 4T1 tumor-bearing mice were treated with control liposomes or clodronate-containing liposomes until IVI-MP was performed. Representative images are shown as in (D). ( F ) Quantification of SHG (cyan) signal intensity in 100 μm Z-stacks of tumors in treated animals. Data are mean ± SEM of measurements from 40-61 Z-stacks from at least 3 different tumors for each treatment group. * P < 0.016. All data are mean ± SEM.

Article Snippet: Anti-human SerpinE2 Ab (MAB2980) was from R&D systems.

Techniques: Staining, Encapsulation, Imaging, Labeling, Control, Liposomes

Journal: Oncotarget

Article Title: Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

doi: 10.18632/oncotarget.12927

Figure Lengend Snippet: Conditioned medium (CM) from 4T1 and 168FARN ( A ) or MDA-MB435 ( B ) cultures was incubated overnight with Ab11, then bound serpinE2 was pulled-down with Protein G, and analyzed by western blot with the rodent-specific antibody, 4B3 (A), or a human-specific antibody (B). (A) The right panel shows a short exposure of serpinE2 complexes from 4T1 CM, but none in 168FARN CM (long exposure on the left), as well as an IP of the preformed tPA/serpinE2 complex. ( C ) SFM loaded with purified human serpinE2 or the preformed serpinE2/tPA complex was IP'd with Ab11 as in (A) & (B) and analyzed by western blot with a human serpinE2-specific antibody. The serpinE2 complex and uncomplexed serpinE2 are indicated with arrowheads.

Article Snippet: Anti-human SerpinE2 Ab (MAB2980) was from R&D systems.

Techniques: Incubation, Western Blot, Purification

Journal: Oncotarget

Article Title: Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

doi: 10.18632/oncotarget.12927

Figure Lengend Snippet: The LRP1 receptor is expressed on tumor cells and on macrophages and we propose that serpinE2 targeting with Ab11 impacts on LRP1 signaling in both cell types. ( A ) In metastatic tumors, serpinE2-LRP1 binding stimulates ERK signaling and secretion of CCL2. Tumor cells display hyperactivation of receptor tyrosine kinases (RTKs); FGFRs, which are active in the 4T1 model, also stimulate ERK pathway activation. We speculate that in macrophages, the combination of high CCL2 levels and serpinE2-activated LRP1 promotes the M2 phenotype. Indeed, there are high levels of phagocytic, Texas-red positive M2 tumor associated macrophages (TAMs) in the metastatic tumors; they are known to be responsible for degrading the matrix during tumor development. ( B ) Ab11 or sepinE2 KD (not drawn in model) lowers ERK pathway activity and the secretion of CCL2, and stimulates TIMP1 secretion. Tyrosine kinase inhibitors (TKIs) block RTK signaling. The matrix-degrading M2 TAMs are decreased in Ab11-treated tumors. Moreover, depleting macrophages with clodronate liposomes results in deposition of a dense collagen matrix, similar to that observed in Ab11-treated tumors. Thus, we propose that the drop in CCL2, which contributes to a decrease in M2 TAMs, as well as blocking serpinE2, which skews the TAMs towards the M1 phenotype, causes the emergence of a dense collagen matrix that inhibits intravasation and metastatic dissemination.

Article Snippet: Anti-human SerpinE2 Ab (MAB2980) was from R&D systems.

Techniques: Binding Assay, Activation Assay, Activity Assay, Blocking Assay, Liposomes

Journal: Oncogene

Article Title: A slow-cycling subpopulation of melanoma cells with highly invasive properties

doi: 10.1038/onc.2017.341

Figure Lengend Snippet: Label-retaining, disseminated melanoma cells express high levels of SerpinE2. ( a ) Table lists proteins identified after whole-cell proteomic analyses of LRC and non-LRC. The last column lists the cell lines with differential expression of the indicated protein. ( b ) Violin plots (left panels) illustrate quantification of SerpinE2 (white spot, top) and BMP1 (white spots, bottom) as determined by mRNA FISH. mRNA counts for single cells are shown on the Y -axes. Note differences in Y -axes. Representative photomicrographs are shown on the right (total magnification: 100 ×). ( c ) Protein expression was analyzed by immunofluorescence and quantified at single-cell levels (scatter plots on left and representative images on right). LRC (red), non-LRC (orange), total cells (gray). ( d ) Flow cytometry of disseminated WM989 melanoma cells detected in three mouse organs (top panel). The percentage of cells double positive for CD146 and SerpinE2 is shown in the upper right quadrants; the lower right quadrants indicate the percentage of cells singly positive for CD146. Micrographs show examples of disseminated SerpinE2-positive cells (arrows, middle and right panels) in mouse lungs. Left panel: negative control.

Article Snippet: Formalin-fixed, paraffin-embedded tissue sections (1–2 μm) were stained with mouse anti-human SerpinE2 antibody (Origene) followed by polymer staining and Fast-Red for 7 min at RT.

Techniques: Quantitative Proteomics, Expressing, Immunofluorescence, Flow Cytometry, Negative Control

Journal: Oncogene

Article Title: A slow-cycling subpopulation of melanoma cells with highly invasive properties

doi: 10.1038/onc.2017.341

Figure Lengend Snippet: SerpinE2 drives melanoma invasiveness. ( a ) Boyden chamber invasion assay of sorted label-retaining cells (LRC) or non-LRC in presence (blue columns) or absence (gray columns) of human recombinant SerpinE2. Bars show fold change in invasion (mean±s.e.m.) of samples with SerpinE2 over untreated cells. ( b ) Invasion assay in presence of an anti-human SerpinE2 neutralizing antibody or Isotype control (ctrl). The percentage of invading cells after neutralization is shown. Data represent mean±s.e.m. of three independent experiments. ( c ) Invasion assay after SerpinE2 knockdown. Data shown are percentage of invading cells found after silencing with 5 different shRNA (Sh_13-17) relative to Sh_ctrl (ctrl). Bars represent mean±s.e.m.

Article Snippet: Formalin-fixed, paraffin-embedded tissue sections (1–2 μm) were stained with mouse anti-human SerpinE2 antibody (Origene) followed by polymer staining and Fast-Red for 7 min at RT.

Techniques: Invasion Assay, Recombinant, Control, Neutralization, Knockdown, shRNA

Journal: Oncogene

Article Title: A slow-cycling subpopulation of melanoma cells with highly invasive properties

doi: 10.1038/onc.2017.341

Figure Lengend Snippet: SerpinE2 expression is increased in malignant cells and correlates with tumor progression. ( a ) Western blot analysis of SerpinE2 secretion by melanoma cells ( n =21) and human melanocytes ( n =5) into culture supernatants. Recombinant SerpinE2 protein (500 ng) was used as a positive control and Ponceau’s staining is shown for loading control. Graph shows quantification (right panel). ( b ) SerpinE2 staining of 3D skin reconstructs with melanocytes (left) and melanoma cells (right). ( c ) GEM_1375 data set analysis of SerpinE2 mRNA expression in melanomas, non-malignant nevi and normal skin. ( d ) Examples of immune histochemistry at two different magnifications of: normal skin, benign nevi, in situ , vertical growth phase (VGP) and lymph node metastatic melanomas ( n =15 for each group). SerpinE2 protein expression is indicated by purple staining. Arrows indicate the dotted SerpinE2 expression pattern found in benign nevi only. ( e ) Intensity score of SerpinE2 expression in normal skin, benign nevi and malignant melanomas (radial growth phase (RGP), VGP and metastatic). Bars represent mean±SEM of tissue sections of 15 specimens from each group; P- value after t -student test are given.

Article Snippet: Formalin-fixed, paraffin-embedded tissue sections (1–2 μm) were stained with mouse anti-human SerpinE2 antibody (Origene) followed by polymer staining and Fast-Red for 7 min at RT.

Techniques: Expressing, Western Blot, Recombinant, Positive Control, Staining, Control, In Situ