Journal: mBio

Article Title: Entamoeba histolytica -Induced Mucin Exocytosis Is Mediated by VAMP8 and Is Critical in Mucosal Innate Host Defense

doi: 10.1128/mBio.01323-17

Figure Lengend Snippet: VAMP8 facilitates mucin exocytosis and is specifically activated by E. histolytica in vitro . (A) shRNA lentivirus-inducible LS174T cells were placed in contact with E. histolytica ( Eh ) for 2 h and [ 3 H]glucosamine-labeled mucin secretion quantified. VAMP8KD cells were compared to uninduced cells, where less mucin secretion was seen in VAMP8KD cells. (B) PKC inhibition by the broad PKC inhibitor bisindolylmaleimide I (BIM1) was hindered in both uninduced and VAMP8KD LS174T cells. (C) shRNA KD of VAMP8 was confirmed by Western blotting, where LS174T and uninduced VAMP8 cells displayed normal VAMP8 expression, in contrast to doxycycline (DOX; 2 µg/ml)-induced VAMP8KD cells, which had >60% less VAMP8. The expression of other SNAREs was not affected by VAMP8KD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) VAMP8 exocytosis was monitored by live-cell imaging using pHluorin, where the area colocalizing to MUC2 showed a time-dependent increase in GFP signal following infection with E. histolytica . Representative tracing from one ROI is displayed. AU, arbitrary units. (E) Indicative of classical exocytosis, VAMP8 translocated to lipid raft domains within the plasma membrane similarly to the mucin secretagogue PMA following infection with E. histolytica . ***, P < 0.001.

Article Snippet: The LS174T cells were passaged with 0.25% trypsin-EDTA (Thermo) once cells reached 90% confluence.

Techniques: In Vitro, shRNA, Labeling, Inhibition, Western Blot, Expressing, Live Cell Imaging, Infection, Clinical Proteomics, Membrane

Journal: mBio

Article Title: Entamoeba histolytica -Induced Mucin Exocytosis Is Mediated by VAMP8 and Is Critical in Mucosal Innate Host Defense

doi: 10.1128/mBio.01323-17

Figure Lengend Snippet: VAMP8KD increases E. histolytica contact with goblet cells due to aberrant mucin exocytosis. (A) LS174T cells were labeled with CFSE and infected with APC-Cellmask-labeled E. histolytica for 30 min, washed with PBS to eliminate nonadherent E. histolytica , dissociated using trypsin, and analyzed by flow cytometry. VAMP8KD cells expressed levels of TurboRFP that were directly proportional to the degree of gene silencing, where 40% of cells were knocked down. APC- E. histolytica scattered very differently, allowing quantification of E. histolytica with LS174T cells. (B) Uninduced and VAMP8KD cells were infected with E. histolytica , and the total amount of E. histolytica was analyzed by the use of APC-CellMask. (C) The multiplicity of infection (MOI) was higher in VAMP8KD cells as quantified by analysis of the ratio of the total amount of E. histolytica remaining after washing to the total amount of LS174T cells. (D and E) Uninduced LS174T cells (D) had less E. histolytica direct binding than VAMP8KD cells (E). Additionally, Q 2 had more E. histolytica bound in TurboRFP + VAMP8KD cells than in uninduced cells in the same well (Q1). (F) E. histolytica direct binding to TurboRFP + VAMP8KD cells was also higher than in uninduced cells. *, P < 0.05.

Article Snippet: The LS174T cells were passaged with 0.25% trypsin-EDTA (Thermo) once cells reached 90% confluence.

Techniques: Labeling, Infection, Flow Cytometry, Binding Assay

Journal: mBio

Article Title: Entamoeba histolytica -Induced Mucin Exocytosis Is Mediated by VAMP8 and Is Critical in Mucosal Innate Host Defense

doi: 10.1128/mBio.01323-17

Figure Lengend Snippet: Lack of VAMP8 allows E. histolytica cytopathic effect and induced apoptosis. (A) LS174T cells were infected with E. histolytica , and the cytopathic effect was assessed by methylene blue staining after 4 h. VAMP8KD cells showed increased cytopathic killing of the monolayer compared to uninduced cells. (B) VAMP8 and classical apoptosis markers were assessed by Western blotting at various time points of infection with E. histolytica . VAMP8 expression was absent in induced cells; however, levels of cleaved caspase 3 (Casp3), caspase 9 (Casp9), and PARP were increased in a time-dependent manner with no spontaneous apoptosis detected in uninfected cells. C, control. (C) There were increased levels of cleaved caspase 3 and 9 in E. histolytica -infected Vamp8 −/− colonic loops, whereas cleavage was absent in Vamp8 +/+ colonic loops. (D) Densitometry was performed to assess increased apoptosis in Vamp8 −/− loops. ***, P < 0.001; *, P < 0.05; NS, not significant.

Article Snippet: The LS174T cells were passaged with 0.25% trypsin-EDTA (Thermo) once cells reached 90% confluence.

Techniques: Infection, Staining, Western Blot, Expressing, Control

Journal: Bioengineered

Article Title: Long non-coding RNA LINC00997 silencing inhibits the progression and metastasis of colorectal cancer by sponging miR-512-3p.

doi: 10.1080/21655979.2021.1882164

Figure Lengend Snippet: Figure 3. LINC00997 silencing suppressed the migration and invasion of CRC cells. (a) LINC00997 level in several CRC cell lines was detected using RT-qPCR. **P < 0.01, ***P < 0.001 vs. HIEC. (b) The level of LINC00997 was tested with RT-qPCR after transfection with shRNA-LINC00997 into HCT116 cells. **P < 0.01, ***P < 0.001. (c and d) Cell migration was tested using wound healing assay. (e and f) The invasive ability of HCT116 cells was determined with Transwell assay. (g) Western blot analysis was used to examine the expression of MMP2 and MMP7. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Several human CRC cell lines (SW480, SW1417, SW620, Caco2, LS174T, HCT15, LoVo, HT29 and HCT116) and the normal intestinal epithelial cell line (HIEC) used in this study were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Quantitative RT-PCR, Transfection, shRNA, Wound Healing Assay, Transwell Assay, Western Blot, Expressing

Journal: Glycobiology

Article Title: Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.

doi: 10.1093/glycob/11.8.633

Figure Lengend Snippet: Fig. 7. Subcellular fractionation of HeLa (A–C) and LS 174T (D–F) cells for assay of glycosyltransferase relocalization on NH4Cl treatment. Cells were homogenized in buffered KCl by passage through syringe needles, debris was pelleted, and the obtained postnuclear supernatant was fractionated by ultracentrifugation on a linear Nycodenz gradient. The nine fractions, recovered from the top to the bottom, were subjected to tabletop ultracentrifugation, and the obtained pellets were reduced and analyzed by western blots of SDS–PAGE, assaying calnexin (A and D), GalNAc-T2/VSV-G (B), endogenous GalNAc-T2 (E) and Gal-T1 (C and F), and developed by the ECL method. In addition to images of the western blots, where the upper in every pair is from control cells and the lower from NH4Cl-treated cells, video densitometry measurements are shown. Open circles, control cells; filled-in circles, NH4Cl-treated cells. The difference in calnexin staining between control and treated cells in A, fraction 8, is not significant.

Article Snippet: The MUC2-producing colon adenocarcinoma cell line LS 174T (ATCC CL 188) (Asker et al., 1995), and monolayer HeLa cells (ATCC CCL 185), stable expressing VSV-Gtagged GalNAc-T2 (Röttger et al., 1998), myc-tagged NAGT I (Nilsson et al., 1993), and GFP-tagged KDEL receptor, were cultivated as described.

Techniques: Fractionation, Western Blot, SDS Page, Control, Staining

Journal: Glycobiology

Article Title: Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.

doi: 10.1093/glycob/11.8.633

Figure Lengend Snippet: Fig. 8. Effect of NH4Cl treatment on the Golgi structure in LS 174T cells. Immuno-EM of ultrathin cryosections of control cells (A) and cells treated with 25 mM NH4Cl for 40 h (B). Bar, 200 nm.

Article Snippet: The MUC2-producing colon adenocarcinoma cell line LS 174T (ATCC CL 188) (Asker et al., 1995), and monolayer HeLa cells (ATCC CCL 185), stable expressing VSV-Gtagged GalNAc-T2 (Röttger et al., 1998), myc-tagged NAGT I (Nilsson et al., 1993), and GFP-tagged KDEL receptor, were cultivated as described.

Techniques: Control

Journal: Glycobiology

Article Title: Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.

doi: 10.1093/glycob/11.8.633

Figure Lengend Snippet: Fig. 9. Gas chromatograms of released mucin oligosaccharides. O-linked oligosaccharides were released from purified insoluble mucins (MUC2) of LS 174T cells by β-elimination and fractionated into neutral, sialylated, and sulfated fractions. Gas chromatograms of permethylated neutral (A and C) and sialylated (B and D) oligosaccharides released from mucins of control (A and B) and NH4Cl-treated (C and D) cells. Designations on the peaks refer to Figure 10.

Article Snippet: The MUC2-producing colon adenocarcinoma cell line LS 174T (ATCC CL 188) (Asker et al., 1995), and monolayer HeLa cells (ATCC CCL 185), stable expressing VSV-Gtagged GalNAc-T2 (Röttger et al., 1998), myc-tagged NAGT I (Nilsson et al., 1993), and GFP-tagged KDEL receptor, were cultivated as described.

Techniques: Purification, Control

Journal: Cancer Management and Research

Article Title:

Apolipoprotein C1 (APOC1) promotes tumor progression via MAPK signaling pathways in colorectal cancer

doi: 10.2147/cmar.s192529

Figure Lengend Snippet: Figure 2 Overexpression of APOC1 mRNA and protein in colorectal cancer (CRC) cell lines. Notes: (A) and (B) Expression of APOC1 protein and mRNA in CRC cell lines (SW1116, SW620, HCT116, SW480 and LS174T) and an immortalized colonic epithelial cell line (NCW460) were examined by Western blotting and qPCR. The expression levels were normalized against GAPDH. The error bars represent the the mean (SD) calculated from three parallel experiments. *P<0.05. (C) and (D) Successful knockdown of APOC1 in SW480 and SW620 CRC cell lines. qPCR and Western blotting were used to detect APOC1 knockdown efficiency in untransfected cells, cells transfected with si-Control (negative control siRNA) and cells transfected with si-RNA1, si-RNA2 and si-RNA3. **P<0.01 and ***P<0.001.

Article Snippet: 50 .2 50 .7 7 on 2 0- Ju n- 20 19 F or p er so na l u se o nl y. Powered by TCPDF (www.tcpdf.org) 1 / 1 Cell culture and treatment Human CRC cell lines (SW1116, SW620, HCT116, SW480 and LS174T) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Over Expression, Expressing, Western Blot, Knockdown, Transfection, Control, Negative Control

Journal: Cancer Management and Research

Article Title:

Apolipoprotein C1 (APOC1) promotes tumor progression via MAPK signaling pathways in colorectal cancer

doi: 10.2147/cmar.s192529

Figure Lengend Snippet: Figure 3 Effects of APOC1 knockdown on the proliferation, apoptosis, migration and invasion of CRC cells after transfection with si-Control or si-APOC1. Notes: (A) Cell proliferation of SW480 and SW620 cells was detected using the CCK-8 assay. (B) Representative images of the clonogenic cell survival assays that were performed in SW480 and SW620 cells. (C) Flow cytometry analysis of the proportion of the cells indicated in each stage of the cell cycle (G1, G2 and S) and the percentage of cells in the G0/G1, S and G2/M phase of the cell cycle were quantified and expressed as the mean ± SD from three independent experiments. (D) Levels of apoptosis in the SW480 and SW620 cells after transfection with si-Control or si-APOC1 for 24 hrs. The cells were stained with Annexin V and PI. (E) Cell migration and invasion in SW480 and SW620 cells were assessed using Transwell assay with or without a Matrigel-coated transwell chamber. Magnification, ×100. (F) Detection of Bcl-2, caspase-9, cyclin D1 and cyclin B1 protein levels by Western blotting. Detection of GAPDH was used as a loading control. Blank: wild-type cells; si-Control: negative control siRNA; si- APOC1: APOC1-siRNA transfected cells (*P<0.05, **P<0.01 Vs si-APOC1).

Article Snippet: 50 .2 50 .7 7 on 2 0- Ju n- 20 19 F or p er so na l u se o nl y. Powered by TCPDF (www.tcpdf.org) 1 / 1 Cell culture and treatment Human CRC cell lines (SW1116, SW620, HCT116, SW480 and LS174T) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Migration, Transfection, Control, CCK-8 Assay, Flow Cytometry, Staining, Transwell Assay, Western Blot, Negative Control