Journal: Journal of medical virology

Article Title: SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1 and NRP-1 in human cells from respiratory, urinary, digestive, reproductive and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: Blocking of infection by SARS‐CoV‐2 and SARS‐CoV pseudoviruses with S1, RBD, and other recombinant proteins of entry‐related factors in Huh‐7 cells. (A−E) Blocking of infection of SARS‐CoV‐2 and SARS‐CoV pseudoviruses by S1 (A), RBD (B), ACE2 (C), Kim‐1 (D), and NRP‐1 (E) recombinant proteins. ANOVA analysis was performed if multiple samples were involved. p‐value was calculated by unpaired two‐tailed Student's t test between uninfected cells and untreated infected cells, and between treated group and untreated infected group. p < 0.05 was considered as statistically significant. ANOVA, analysis of variance; RBD, receptor‐binding domain; Kim‐1, kidney injury molecule‐1; NRP‐1, neuropilin‐1; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SARS‐CoV, severe acute respiratory syndrome coronavirus

Article Snippet: Table 1 Cell name Origin Source of cell Culture medium Respiratory system JHU‐029 Laryngeal squamous cell carcinoma CVCL_5993 RPMI‐1640 complete medium NCL H460 Large cell lung cancer ATCC HTB‐177 RPMI‐1640 complete medium NCL H322 Large cell lung cancer ATCC CRL­5806 DMEM complete medium NCL H520 Large cell lung cancer ATCC HTB‐182 DMEM complete medium A549 Lung adenocarcinoma ATCC CRM‐CCL‐185 DMEM complete medium Primary human lobar bronchial epithelial cells (HLBEC) Lobar bronchial tissue Lifeline® Cell Technology Lifeline® BronchiaLife™ Medium Primary human small airway epithelial cells (HSAEC) Small airway tissue Lifeline® Cell Technology Lifeline® BronchiaLife™ Medium Urinary system 769‐P Renal cell adenocarcinoma ATCC CRL‐1933 RPMI‐1640 complete medium 768‐O Renal cell adenocarcinoma ATCC CRL‐1932 RPMI‐1640 complete medium A498 Renal cell adenocarcinoma ATCC HTB‐44 RPMI‐1640 complete medium Caki‐1 Renal cell carcinoma ATCC HTB‐46 DMEM complete medium ACHN Renal cell adenocarcinoma ATCC CRL‐1611 DMEM complete medium HRC45 Renal cell carcinoma CVCL_IS24 DMEM complete medium HRC63 Renal cell carcinoma CVCL_IS25 DMEM complete medium HRC59 Renal cell carcinoma FCCC DMEM complete medium Immune system BCP‐1 Primary effusion lymphoma ATCC CRL‐2294 RPMI‐1640 complete medium BC‐3 Primary effusion lymphoma ATCC CRL‐2277 RPMI‐1640 complete medium BJAB Primary effusion lymphoma CVCL_5711 RPMI‐1640 complete medium THP‐1 Acute monocytic leukemia ATCC TIB‐202 RPMI‐1640 complete medium Digestive system Huh‐7 Hepatocellular carcinoma cell JCRB0403 DMEM complete medium PCI‐13 Oral cavity squamous cell carcinoma CVCL_C182 RPMI‐1640 complete medium UD‐SCC‐2 Hypopharyngeal squamous cell carcinoma CVCL_E325 RPMI‐1640 complete medium Reproductive system HUVEC Umbilical Vein Endothelial Cells ATCC PCS‐100‐010 ECBM(Cell applications, 210‐490) T47D Ductal carcinoma ATCC HTB‐133 RPMI‐1640 complete medium MCF‐7 Breast adenocarcinoma ATCC HTB‐22 RPMI‐1640 complete medium Open in a separate window Note : DMEM or RPMI‐1640 complete medium is made of DMEM or RPMI‐1640 basal medium with 10% fetal bovine serum (FBS) and 1% Penicillin‐Streptomycin 100× Solution (25‐512, GenClone).

Techniques: Blocking Assay, Infection, Recombinant, Two Tailed Test, Binding Assay

Journal: Journal of medical virology

Article Title: SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1 and NRP-1 in human cells from respiratory, urinary, digestive, reproductive and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: Expression levels of ACE2, TMPRSS2, Kim‐1, and NRP‐1 proteins in different cell lines/types, and examination of ACE glycosylation in Huh‐7 cells analyzed by Western blot analysis. (A) Expression levels of ACE2, TMPRSS2, Kim‐1, and NRP‐1 proteins in different cell lines/types. Results showed that there are different expression profiles of ACE2, TMPRSS2, Kim‐1, and NRP‐1 in different cell lines/types. (B) Examination of ACE glycosylation in Huh‐7 cells. Untreated and PNGase F‐treated cell lysates were examined. β‐tubulin was used for loading normalization. Two bands of ~85 kD and ~120 kD were detected for ACE2 protein representing the unglycosylated and glycosylated forms of ACE2 (ungly‐ACE2 and gly‐ACE2), respectively. Kim‐1, kidney injury molecule‐1; NRP‐1, neuropilin‐1; TMPRSS2, transmembrane serine protease 2

Article Snippet: Table 1 Cell name Origin Source of cell Culture medium Respiratory system JHU‐029 Laryngeal squamous cell carcinoma CVCL_5993 RPMI‐1640 complete medium NCL H460 Large cell lung cancer ATCC HTB‐177 RPMI‐1640 complete medium NCL H322 Large cell lung cancer ATCC CRL­5806 DMEM complete medium NCL H520 Large cell lung cancer ATCC HTB‐182 DMEM complete medium A549 Lung adenocarcinoma ATCC CRM‐CCL‐185 DMEM complete medium Primary human lobar bronchial epithelial cells (HLBEC) Lobar bronchial tissue Lifeline® Cell Technology Lifeline® BronchiaLife™ Medium Primary human small airway epithelial cells (HSAEC) Small airway tissue Lifeline® Cell Technology Lifeline® BronchiaLife™ Medium Urinary system 769‐P Renal cell adenocarcinoma ATCC CRL‐1933 RPMI‐1640 complete medium 768‐O Renal cell adenocarcinoma ATCC CRL‐1932 RPMI‐1640 complete medium A498 Renal cell adenocarcinoma ATCC HTB‐44 RPMI‐1640 complete medium Caki‐1 Renal cell carcinoma ATCC HTB‐46 DMEM complete medium ACHN Renal cell adenocarcinoma ATCC CRL‐1611 DMEM complete medium HRC45 Renal cell carcinoma CVCL_IS24 DMEM complete medium HRC63 Renal cell carcinoma CVCL_IS25 DMEM complete medium HRC59 Renal cell carcinoma FCCC DMEM complete medium Immune system BCP‐1 Primary effusion lymphoma ATCC CRL‐2294 RPMI‐1640 complete medium BC‐3 Primary effusion lymphoma ATCC CRL‐2277 RPMI‐1640 complete medium BJAB Primary effusion lymphoma CVCL_5711 RPMI‐1640 complete medium THP‐1 Acute monocytic leukemia ATCC TIB‐202 RPMI‐1640 complete medium Digestive system Huh‐7 Hepatocellular carcinoma cell JCRB0403 DMEM complete medium PCI‐13 Oral cavity squamous cell carcinoma CVCL_C182 RPMI‐1640 complete medium UD‐SCC‐2 Hypopharyngeal squamous cell carcinoma CVCL_E325 RPMI‐1640 complete medium Reproductive system HUVEC Umbilical Vein Endothelial Cells ATCC PCS‐100‐010 ECBM(Cell applications, 210‐490) T47D Ductal carcinoma ATCC HTB‐133 RPMI‐1640 complete medium MCF‐7 Breast adenocarcinoma ATCC HTB‐22 RPMI‐1640 complete medium Open in a separate window Note : DMEM or RPMI‐1640 complete medium is made of DMEM or RPMI‐1640 basal medium with 10% fetal bovine serum (FBS) and 1% Penicillin‐Streptomycin 100× Solution (25‐512, GenClone).

Techniques: Expressing, Western Blot

Journal: Bioscience Reports

Article Title: LncRNA GNAS-AS1 facilitates ER + breast cancer cells progression by promoting M2 macrophage polarization via regulating miR-433-3p/GATA3 axis

doi: 10.1042/BSR20200626

Figure Lengend Snippet: ( A ) The relative quantity of GNAS-AS1 in ER + breast cancer tissues and adjacent normal tissues was examined by qRT-PCR. ( B ) Human monocytes were isolated from PBMCs with antibody against CD14 and CD11b and analyzed using flow cytometry. ( C ) Flow cytometry was used to quantify the proportion of M1 or M2 macrophages. ( D ) M1 macrophage markers (TNF-α, IL-6) were measured by qRT-PCR. ( E ) M2 macrophage markers (IL-10, Arginase-1) were detected by qRT-PCR. ( F ) qRT-PCR was performed to determine GNAS-AS1 expression in TAMs (M0, M1, M2). ( G ) The expression level of GNAS-AS1 in breast cancer cells (T47D and MCF-7) and normal mammary epithelial cells (MCF10A) were determined by qRT-PCR. Data with error bars are presented as the mean ± SD; * P <0.05, ** P <0.01, *** P <0.001 as determined by the Student’s t test or one-way ANOVA test.

Article Snippet: The cells were maintained in RPMI 1640 medium (THP-1) (Hyclone, America), MEGM medium (MCF10A) (Hyclone, America) and DMEM/F-12 (T47D, MCF-7) (Gibco, America), supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, America) and 1% penicillin/streptomycin (Beyotime Biotechnology, China).

Techniques: Quantitative RT-PCR, Isolation, Flow Cytometry, Expressing

Journal: Bioscience Reports

Article Title: LncRNA GNAS-AS1 facilitates ER + breast cancer cells progression by promoting M2 macrophage polarization via regulating miR-433-3p/GATA3 axis

doi: 10.1042/BSR20200626

Figure Lengend Snippet: In IL-4 treated THP-1-differentiated macrophages, pSin-GNAS-AS1 plasmids or negative plasmids were transfected into cells, then next experiments were conducted. ( A ) The level of GNAS-AS1 was evaluated using qRT-PCR. ( B ) Flow cytometry was used to quantify the proportion of M1 or M2 macrophages. ( C ) qRT-PCR was performed to determine the expression levels of M2 macrophage markers (IL-10, Arginase-1). ( D ) CCK-8 assay was conducted to assess the cell viability of T47D and MCF-7 cells co-cultured with above treated THP-1-differentiated macrophages. ( E ) Wound healing assay was used to examine the migration of T47D and MCF-7 cells co-cultured with above treated THP-1-differentiated macrophages. ( F ) Transwell was applied to detect the invasion of T47D and MCF-7 cells co-cultured with above treated THP-1-differentiated macrophages. Data with error bars are presented as the mean ± SD. The Student’s t test and one-way ANOVA test were used to determine significance; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The cells were maintained in RPMI 1640 medium (THP-1) (Hyclone, America), MEGM medium (MCF10A) (Hyclone, America) and DMEM/F-12 (T47D, MCF-7) (Gibco, America), supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, America) and 1% penicillin/streptomycin (Beyotime Biotechnology, China).

Techniques: Transfection, Quantitative RT-PCR, Flow Cytometry, Expressing, CCK-8 Assay, Cell Culture, Wound Healing Assay, Migration

Journal: Bioscience Reports

Article Title: LncRNA GNAS-AS1 facilitates ER + breast cancer cells progression by promoting M2 macrophage polarization via regulating miR-433-3p/GATA3 axis

doi: 10.1042/BSR20200626

Figure Lengend Snippet: ( A ) The binding site of miR-433-3p on GNAS-AS1 3′-UTR region. ( B ) Dual luciferase reporter assay was used to validate the molecular relationship between miR-433-3p and GNAS-AS1 in THP-1-differentiated macrophages. ( C ) qRT-PCR was performed to assess the expression of GNAS-AS1 in THP-1-differentiated macrophages transfected with pSin-GNAS-AS1 plasmids or si-GNAS-AS1. ( D ) qRT-PCR was performed to assess the expression of miR-433-3p in THP-1-differentiated macrophages transfected with pSin-GNAS-AS1 plasmids or si-GNAS-AS1. Data with error bars are presented as the mean ± SD. The Student’s t test and one-way ANOVA test were used to determine significance; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The cells were maintained in RPMI 1640 medium (THP-1) (Hyclone, America), MEGM medium (MCF10A) (Hyclone, America) and DMEM/F-12 (T47D, MCF-7) (Gibco, America), supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, America) and 1% penicillin/streptomycin (Beyotime Biotechnology, China).

Techniques: Binding Assay, Luciferase, Reporter Assay, Quantitative RT-PCR, Expressing, Transfection

Journal: Bioscience Reports

Article Title: LncRNA GNAS-AS1 facilitates ER + breast cancer cells progression by promoting M2 macrophage polarization via regulating miR-433-3p/GATA3 axis

doi: 10.1042/BSR20200626

Figure Lengend Snippet: In IL-4 treated THP-1-differentiated macrophages, pSin-GNAS-AS1 plasmids were co-transfected with/without miR-433-3p mimics, then next experiments were conducted. ( A ) qRT-PCR was performed to analyze the expression of miR-433-3p. ( B ) Flow cytometry was used to quantify the proportion of M1 or M2 macrophages. ( C ) qRT-PCR was applied to determine the expressions of M2 macrophage markers (IL-10, Arginase-1). ( D ) CCK-8 assay was conducted to assess the cell viability of T47D and MCF-7 cells co-cultured with above treated THP-1-differentiated macrophages. ( E ) Wound healing assay was used to detect the migration of T47D and MCF-7 cells co-cultured with above treated THP-1-differentiated macrophages. ( F ) Transwell assay was performed to elevate the invasion of T47D and MCF-7 cells co-cultured with above treated THP-1-differentiated macrophages. Data with error bars are presented as the mean ± SD. The Student’s t test and one-way ANOVA test were used to determine significance; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The cells were maintained in RPMI 1640 medium (THP-1) (Hyclone, America), MEGM medium (MCF10A) (Hyclone, America) and DMEM/F-12 (T47D, MCF-7) (Gibco, America), supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, America) and 1% penicillin/streptomycin (Beyotime Biotechnology, China).

Techniques: Transfection, Quantitative RT-PCR, Expressing, Flow Cytometry, CCK-8 Assay, Cell Culture, Wound Healing Assay, Migration, Transwell Assay

Journal: Bioscience Reports

Article Title: LncRNA GNAS-AS1 facilitates ER + breast cancer cells progression by promoting M2 macrophage polarization via regulating miR-433-3p/GATA3 axis

doi: 10.1042/BSR20200626

Figure Lengend Snippet: ( A ) The binding site of miR-433-3p on GATA3 3′-UTR region. ( B ) Dual luciferase reporter assay was used to validate the binding relationship between miR-433-3p and GATA3 in THP-1-differentiated macrophages. ( C ) qRT-PCR was performed to assess the expression of miR-433-3p in THP-1-differentiated macrophages transfected with miR-433-3p mimics or miR-433-3p inhibitor. ( D ) qRT-PCR was performed to assess the expression of GATA3 in THP-1-differentiated macrophages transfected with miR-433-3p mimics or miR-433-3p inhibitor. Data with error bars are presented as the mean ± SD; The Student’s t test and one-way ANOVA test were used to determine significance; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The cells were maintained in RPMI 1640 medium (THP-1) (Hyclone, America), MEGM medium (MCF10A) (Hyclone, America) and DMEM/F-12 (T47D, MCF-7) (Gibco, America), supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, America) and 1% penicillin/streptomycin (Beyotime Biotechnology, China).

Techniques: Binding Assay, Luciferase, Reporter Assay, Quantitative RT-PCR, Expressing, Transfection

Journal: Bioscience Reports

Article Title: LncRNA GNAS-AS1 facilitates ER + breast cancer cells progression by promoting M2 macrophage polarization via regulating miR-433-3p/GATA3 axis

doi: 10.1042/BSR20200626

Figure Lengend Snippet: ( A ) The protein level of GATA3 was examined by Western blot analysis in THP-1 cells transfected with pSin-GNAS-AS1 plasmids or si-GNAS-AS1. ( B–G ) In IL-4 treated THP-1-differentiated macrophages, pSin-GNAS-AS1 plasmids were co-transfected with/without siGATA3, then, next experiments were performed. (B) The mRNA level of GATA3 was analyzed by qRT-PCR. (C) Flow cytometry was used to quantify the proportion of M2 macrophages polarization. (D) qRT-PCR was performed to determine the expressions of M2 macrophage markers (IL-10, Arginase-1). (E) CCK-8 was used to detect the migration of T47D and MCF-7 cells co-cultured with above treated THP-1-differentiated macrophages. (F) Wound healing assay was used to detect the migration of T47D and MCF-7 cells co-cultured with above treated THP-1-differentiated macrophages. (G) Transwell was used to detect the invasion of T47D and MCF-7 cells co-cultured with above treated THP-1-differentiated macrophages. Data with error bars are presented as the mean ± SD. The Student’s t test and one-way ANOVA test were used to determine significance; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The cells were maintained in RPMI 1640 medium (THP-1) (Hyclone, America), MEGM medium (MCF10A) (Hyclone, America) and DMEM/F-12 (T47D, MCF-7) (Gibco, America), supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, America) and 1% penicillin/streptomycin (Beyotime Biotechnology, China).

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, CCK-8 Assay, Migration, Cell Culture, Wound Healing Assay

Journal: Cancer Science

Article Title: Tumor associated macrophage expressing CD 204 is associated with tumor aggressiveness of esophageal squamous cell carcinoma

doi: 10.1111/cas.12188

Figure Lengend Snippet: Induction of CD204 and VEGF‐A expression in 12‐O‐tetradecanoylphorbol‐l3‐acetate (TPA)‐treated THP‐1 human monocytic cell line by conditioned media of human esophageal cancer cell lines. (a) THP‐1 cells were treated with 200 nM TPA for 2 days to induce macrophage‐like differentiation, then exposed to 50% conditioned media of TE series esophageal cancer cell lines (TECMs) for 2 days. (b–d) Morphological changes of THP‐1 by TPA and TECM treatment. Before treatment, THP‐1 cells were round with diameter around 15 μm and did not attach to the surface of culture dish (b) After treatment with TPA, they adhered to the plate substrate with various cytoplasmic projections (c) They kept attaching with extended cytoplasmic projections with the consecutive exposure to the CM of TE‐8 (d) Bar 20 μm. (e) CD163 mRNA expression was observed in untreated, TPA‐treated and TECM treated THP‐1, while CD204 expression was induced by TECM exposure with TPA‐pretreatment. Reverse transcription‐polymerase chain reaction analysis. Glyceraldehyde 3‐phosphate dehydrogenase was used as a control. (f, g) Quantification of CD163+ (f) and CD204+ (g) THP‐1 cells in culture by immunofluorescence exposed with TECMs after TPA‐treatment. Mean numbers of CD163+ or CD204+ cells were compared with those of TPA‐treated THP‐1 cells. Significant induction of numbers of CD204+ (P < 0.05) but not of CD163+ THP‐1 cells was observed after TECM exposure. Paired t‐test. Bars, mean ± standard error of the mean (SEM). (h) Relative expression levels of VEGF‐A mRNA determined by real time qRT‐PCR in THP‐1 cells exposed with TECMs after TPA‐treatment. Each TECM demonstrated significant induction of VEGF‐A expression (P < 0.05). Paired t‐test. Bars mean ± SEM.

Article Snippet: 25 We routinely maintained ESCC cell lines in RPMI‐1640 (Wako, Osaka, Japan) and THP‐1 cells in DMEM (Wako) with 10% FBS (Sigma‐Aldrich, Tokyo, Japan) and 1% antibiotic–antimycotic (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Quantitative RT-PCR