Journal: Biomedicines

Article Title: Duodenal Adenocarcinoma Is Characterized by Acidity, High Infiltration of Macrophage, and Activated Linc01559–GRSF1 Axis

doi: 10.3390/biomedicines13071611

Figure Lengend Snippet: The microenvironment of duodenal adenocarcinoma is characterized by acidity and a high level of macrophage infiltration. ( a ) Principal component analysis (PCA) of transcriptome sequencing data from 11 paired samples of duodenal adenocarcinoma tumor tissues and adjacent normal tissues; ( b , c ) heatmap of 18 differentially expressed lncRNAs ( b ) and their correlated mRNAs ( c ) between normal and tumor tissues, as described in a Signaling pathways related to these genes are shown in panel C; ( d ) cancer hallmark signaling pathways associated with the genes co-expressed with the differentially expressed lncRNAs; ( e ) infiltration levels of immune cells in normal and duodenal adenocarcinoma tumor tissues; ( f ) mRNA expression levels of acidic environmental markers in normal and duodenal adenocarcinoma tumor tissues; ( g – i ) correlation analysis of expression between 18 lncRNAs and macrophage markers ( g ); macrophage markers and acidic environmental markers ( h ); and 18 lncRNAs and acidic environmental markers ( i ); The Pearson correlation coefficient (r) and p -values are shown * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Human duodenal adenocarcinoma cell lines (WDC-1, HuTu-80) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 10% FBS (Bioexploper, Boulder, CO, USA) and penicillin/streptomycin (Gibco).

Techniques: Sequencing, Protein-Protein interactions, Expressing

Journal: Biomedicines

Article Title: Duodenal Adenocarcinoma Is Characterized by Acidity, High Infiltration of Macrophage, and Activated Linc01559–GRSF1 Axis

doi: 10.3390/biomedicines13071611

Figure Lengend Snippet: The microenvironment of duodenal adenocarcinoma is characterized by acidity and a high level of macrophage infiltration and Linc01559 expression. ( a ) Heatmap of differentially expressed coding genes between normal and duodenal adenocarcinoma tumor tissues; ( b ) the proportion of protein-coding genes, lncRNAs, and processed pseudogenes in duodenal adenocarcinoma tumor tissues; ( c ) qPCR of RNA expression of differentially expressed coding genes as described in ( a ); ( d ) infiltration levels of immune cells in duodenal adenocarcinoma tumor tissues; ( e ) infiltration levels of macrophages in normal and duodenal adenocarcinoma tumor tissues; ( f ) correlation analysis of the expression between Linc01559 and macrophages; and ( g ) correlation analysis of the expression between Linc01559 and acidic environmental markers. Three independent experiments were performed to obtain the data in ( c ). The data are shown as the mean ± SD; the Pearson correlation coefficient (r) and p -values are shown.

Article Snippet: Human duodenal adenocarcinoma cell lines (WDC-1, HuTu-80) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 10% FBS (Bioexploper, Boulder, CO, USA) and penicillin/streptomycin (Gibco).

Techniques: Expressing, RNA Expression

Journal: Biomedicines

Article Title: Duodenal Adenocarcinoma Is Characterized by Acidity, High Infiltration of Macrophage, and Activated Linc01559–GRSF1 Axis

doi: 10.3390/biomedicines13071611

Figure Lengend Snippet: High expression of Linc01559 is positively correlated with an acidic environment and the STAT3-cMYC pathway in duodenal adenocarcinoma. ( a ) The volcano plot shows significantly overexpressed lncRNAs in duodenal adenocarcinoma tumor tissues; ( b ) qPCR of RNA expression ofdifferentially expressed lncRNAs described in a; ( c ) mRNA expression levels of acidic environmental markers in Linc01559 high and Linc01559 low expression group; ( d – f ) heatmap of genes co-expressed with Linc01559 ( e ) and the cancer hallmark signaling pathways associated with these genes ( d , f ) between normal and tumor tissues are shown; ( g ) mRNA expression of HIF1α, STAT3 and c-MYC in normal and tumor tissues; ( h ) correlation analysis of the expression between Linc01559 and HIF1α, STAT3, c-MYC. Three independent experiments were performed to obtain the data in ( b ); The data are shown as the mean ± SD; the Pearson correlation coefficient (r) and p -values are shown * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human duodenal adenocarcinoma cell lines (WDC-1, HuTu-80) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 10% FBS (Bioexploper, Boulder, CO, USA) and penicillin/streptomycin (Gibco).

Techniques: Expressing, RNA Expression, Protein-Protein interactions

Journal: Biomedicines

Article Title: Duodenal Adenocarcinoma Is Characterized by Acidity, High Infiltration of Macrophage, and Activated Linc01559–GRSF1 Axis

doi: 10.3390/biomedicines13071611

Figure Lengend Snippet: ( a ) Correlation analysis among Linc01559 , IL13, HIF1A, c-MYC, and STAT3 in normal and tumor tissues; ( b , c ) correlation analysis among Linc01559 ( b ), GRSF1 ( c ), and stemness markers; ( d ) mRNA expression levels of GRSF1 in normal and duodenal adenocarcinoma tumor tissues; ( e ) correlation analysis of the expression between GRSF1 and acidic environmental markers CA9, LDHA in tumor tissues.; and ( f ) representative images of tumor spheres (larger than 50 µm in diameter) formed after knockdown of GRSF1 in WDC-1 duodenal cancer cells under acidic and alkaline conditions. Scale bar: 100 µm. The Pearson correlation coefficient (r) and p -values are shown.

Article Snippet: Human duodenal adenocarcinoma cell lines (WDC-1, HuTu-80) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 10% FBS (Bioexploper, Boulder, CO, USA) and penicillin/streptomycin (Gibco).

Techniques: Expressing, Knockdown

Journal: Biomedicines

Article Title: Duodenal Adenocarcinoma Is Characterized by Acidity, High Infiltration of Macrophage, and Activated Linc01559–GRSF1 Axis

doi: 10.3390/biomedicines13071611

Figure Lengend Snippet: Linc01559 promotes the malignant phenotype of duodenal adenocarcinoma cells. ( a ) qPCR was used to verify the expression of Linc01559 in duodenal adenocarcinoma cells treated with control and Linc01559 -targeting siRNA; ( b , c ) cell viability of control and Linc01559 -knockdown duodenal adenocarcinoma cells is shown, with the IC50 indicated by a dotted line; ( d – f ) representative images ( d ) and quantified data ( e , f ) from the transwell assay demonstrate the migration and invasion ability of control and Linc01559 -knockdown duodenal adenocarcinoma cells. (scale bar,100 µm); ( g , h ) representative images ( g ) and quantified data ( h ) of the colony formation assay illustrate the stemness characteristics of control and Linc01559 -knockdown duodenal adenocarcinoma cells; ( i , j ) representative images ( i ) and quantified data ( j ) from flow cytometry analysis show the apoptosis of control and Linc01559 -knockdown duodenal adenocarcinoma cells; ( k ) correlation analysis between Linc01559 and stemness marker EPCAM, CD44, and SOX2. Three independent experiments were performed to obtain the data in ( a , e , f , h , j ). The data are shown as the mean ± SD; the Pearson correlation coefficient (r) and p -values are shown **** p < 0.0001.

Article Snippet: Human duodenal adenocarcinoma cell lines (WDC-1, HuTu-80) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 10% FBS (Bioexploper, Boulder, CO, USA) and penicillin/streptomycin (Gibco).

Techniques: Expressing, Control, Knockdown, Transwell Assay, Migration, Colony Assay, Flow Cytometry, Marker

Journal: Biomedicines

Article Title: Duodenal Adenocarcinoma Is Characterized by Acidity, High Infiltration of Macrophage, and Activated Linc01559–GRSF1 Axis

doi: 10.3390/biomedicines13071611

Figure Lengend Snippet: GRSF1 is the target protein interacting with Linc01559 . ( a , b ) qPCR analysis of the RNA expression levels of β-actin, U6, and Linc01559 in the nuclear and cytoplasmic fractions of HuTu-80 ( a ) and WDC-1 ( b ) cells; ( c ) translatome DB analysis of the protein expression of lncRNAs as described in a and mRNAs; ( d ) RNA pull down of the detected protein that interacted with Linc01559; ( e ). The secondary structure diagram of Linc01559 ; ( f ) mass spectrometry results as described in d; ( g , h ) the interaction between GRSF1 and Linc01559 is detected by RIP ( g ), the immunoprecipitated RNA is quantified by qPCR(h); ( i ) western blot of the protein expression of GRSF1 in control and Linc01559 -knockdown duodenal adenocarcinoma cells; ( j ) correlation analysis of the expression between Linc01559 and GRSF1. Three independent experiments were performed to obtain the data in ( a , b , h ). The data are shown as the mean ± SD; the Pearson correlation coefficient (r) and p -values are shown *** p < 0.001.

Article Snippet: Human duodenal adenocarcinoma cell lines (WDC-1, HuTu-80) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 10% FBS (Bioexploper, Boulder, CO, USA) and penicillin/streptomycin (Gibco).

Techniques: RNA Expression, Expressing, Mass Spectrometry, Immunoprecipitation, Western Blot, Control, Knockdown

Journal: Biomedicines

Article Title: Duodenal Adenocarcinoma Is Characterized by Acidity, High Infiltration of Macrophage, and Activated Linc01559–GRSF1 Axis

doi: 10.3390/biomedicines13071611

Figure Lengend Snippet: Linc01559 affects the migration and apoptosis of duodenal adenocarcinoma through upregulating GRSF1. ( a , b ) Representative images ( left ) (scale bar, 100 µm) and quantified data ( right ) of the transwell assay showed the migration ability of control and Linc01559 -knockdown duodenal adenocarcinoma cells treated with vector or GRSF1 plasmid; ( c , d ) representative images ( left ) and quantified data ( right ) of the flow cytometry showed the apoptosis of control and Linc01559 -knockdown duodenal adenocarcinoma cells treated with vector or GRSF1 plasmid. Three independent experiments were performed to obtain the data in ( a – d ). The data are shown as the mean ±SD; p -values are shown * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Human duodenal adenocarcinoma cell lines (WDC-1, HuTu-80) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 10% FBS (Bioexploper, Boulder, CO, USA) and penicillin/streptomycin (Gibco).

Techniques: Migration, Transwell Assay, Control, Knockdown, Plasmid Preparation, Flow Cytometry

Journal: Biomedicines

Article Title: Duodenal Adenocarcinoma Is Characterized by Acidity, High Infiltration of Macrophage, and Activated Linc01559–GRSF1 Axis

doi: 10.3390/biomedicines13071611

Figure Lengend Snippet: STAT3/c-MYC pathway accelerates the stemness of duodenal adenocarcinoma via the Linc01559 –GRSF1 signal. ( a ) Correlation analysis among Linc01559, GRSF1, acidic environmental markers LDHA, CA12, and macrophage markers IL1B; ( b ) correlation analysis among GRSF1 and stemness marker EPCAM, CD44, and SOX2; ( c ) western blot of the protein expression of GRSF1, EPCAM, and CD44 in control and GRSF1-knockdown duodenal adenocarcinoma cells; ( d ) tumor spheres (with diameters larger than 50 µm) formed by control and GRSF1-knockdown duodenal adenocarcinoma cells cultured in alkaline and acidic conditions; ( e ) western blot of the protein expression of EPCAM and CD44 in control and Linc01559-knockdown duodenal adenocarcinoma cells treated with vector or GRSF1 plasmid; ( f , g ) western blot of the protein expression of STAT3 or c-MYC in control and STAT3-knockdown or c-MYC-knockdown duodenal adenocarcinoma cells; ( h , i ) qPCR of the RNA expression of Linc01559 in control and STAT3-knockdown or c-MYC-knockdown duodenal adenocarcinoma cells; ( j ) GSEA of the cancer hallmark signals in GRSF1 high and GRSF1 low group from duodenal adenocarcinoma tumor tissues; ( k ) the mRNA expression of stemness markers EPCAM, CD44, NUMB, and EMT markers OCLN, CDH1, and VIM in GRSF1 high and GRSF1 low group. Three independent experiments were performed to obtain the data in ( d , h , i ). The data are shown as the mean ± SD; the Pearson correlation coefficient (r) and p -values are shown * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human duodenal adenocarcinoma cell lines (WDC-1, HuTu-80) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 10% FBS (Bioexploper, Boulder, CO, USA) and penicillin/streptomycin (Gibco).

Techniques: Marker, Western Blot, Expressing, Control, Knockdown, Cell Culture, Plasmid Preparation, RNA Expression

Journal: Genes to Cells

Article Title: Establishment of a Novel Caco‐2‐Based Cell Culture System for Human Sapovirus Propagation

doi: 10.1111/gtc.70007

Figure Lengend Snippet: Susceptibility of various cell lines to HuSaV infection. (A) Changes in HuSaV RNA copy numbers in the culture supernatants of HuTu80 and HEK293T cells following inoculation with a HuSaV GI.1 (AH20)‐positive stool suspension (~4 × 10 7 copies/well in 96‐well plates). Each dot represents individual data points; bars indicate the geometric mean value of the HuSaV RNA copy numbers; error bars denote the geometric standard deviation (SD). This experiment was performed once with five technical replicates. (B) Changes in HuSaV RNA copy numbers in the culture supernatants of Caco‐2, HCT15, HCT116, Caco‐2/Cas9, and C2BBe1 cells following inoculation with a HuSaV GI.1 (AH20)‐positive stool suspension (~2 × 10 6 copies/well in 96‐well plates). Each dot represents individual data points; bars indicate the geometric mean HuSaV RNA copy numbers; error bars denote the geometric SD. This experiment was performed once with five technical replicates. (C) Immunofluorescence staining of the viral protein VP1 in HuTu80, HEK293T, Caco‐2, and Caco‐2/Cas9 cells at 3 dpi with a HuSaV GI.1 (AH20)‐positive stool suspension; upper panels: 4× objective lens, lower panels: 40× objective lens. (D) Flow cytometry analysis of Caco‐2 and Caco‐2/Cas9 cells infected with a HuSaV GI.1 (AH20)‐positive stool suspension at 4 dpi. Blue: VP1‐negative cells (uninfected), red: VP1‐positive cells (infected).

Article Snippet: The human duodenal cancer cell line HuTu80 was purchased from ATCC and cultured in Iscove's Modified Dulbecco's Medium (IMDM; SIGMA) supplemented with 1% GlutaMAX‐I (Gibco, Massachusetts, USA), 1% penicillin/streptomycin (Nacalai Tesque), and 5% heat‐inactivated fetal bovine serum (FBS: CAPRICORN, Ebsdorfergrund, Germany).

Techniques: Infection, Suspension, Standard Deviation, Immunofluorescence, Staining, Flow Cytometry

Journal: Genes to Cells

Article Title: Establishment of a Novel Caco‐2‐Based Cell Culture System for Human Sapovirus Propagation

doi: 10.1111/gtc.70007

Figure Lengend Snippet: Serial passage of HuSaV GI.1 in HuTu80, HEK293T, Caco‐2, and Caco‐2/Cas9 cells. HuSaV RNA copy numbers in the culture supernatants at 0 or 1 dpi (immediately after medium replacement) and 7 dpi for each passage, up to P + 5, in (A) HuTu80, (B) HEK293T, (C) Caco‐2, and (D) Caco‐2/Cas9 cells.

Article Snippet: The human duodenal cancer cell line HuTu80 was purchased from ATCC and cultured in Iscove's Modified Dulbecco's Medium (IMDM; SIGMA) supplemented with 1% GlutaMAX‐I (Gibco, Massachusetts, USA), 1% penicillin/streptomycin (Nacalai Tesque), and 5% heat‐inactivated fetal bovine serum (FBS: CAPRICORN, Ebsdorfergrund, Germany).

Techniques: