colorectal epithelial cell line caco2  (ATCC)

Journal: Toxins

Article Title: The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus : Subunit Interactions in Cell-Free Systems

doi: 10.3390/toxins13110807

Figure Lengend Snippet: Hbl induced membrane perforation monitored by propidium iodide staining. Cell-free synthesized Hbl was assessed for its cytotoxic effect on CaCo2 cells. Upon Hbl induced membrane rupture, the DNA intercalating agent propidium iodide (PI) enters the cell’s cytoplasm. DNA bound PI was detected at 616 nm using the Mithras LB 943 (Berthold). Hbl tripartite toxin was coexpressed and the SN fraction ( a ) and the MF fraction ( b ) were analyzed. An NTC consisting of a volume equivalent reaction without a coding DNA template was used as a negative control. Hbl protein concentrations ranging from 0.01 nM up to 2.5 nM were tested. Standard deviations were calculated from triplicate samples of three independent experiments ( n = 9) with the exception of the MF NTC at 0.25 nM as indicated by * ( n = 5).

Article Snippet: Therefore, the epithelial colorectal adenocarcinoma cell line CaCo2 (ATCC) was cultured in minimum essential medium (MEM) supplemented with 20% fetal calf serum (Merck, Biochrom GmbH, Berlin, Germany), 1% non-essential amino acids (Merck, Biochrom GmbH) and 1% penicillin/streptomycin (Merck, Biochrom GmbH).

Techniques: Membrane, Staining, Synthesized, Negative Control

Journal: Toxins

Article Title: The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus : Subunit Interactions in Cell-Free Systems

doi: 10.3390/toxins13110807

Figure Lengend Snippet: Membrane perforation assay of the individual Hbl subunits. Cell-free synthesized Hbl, the individual subunits as well as two subunits synthesized together were assessed for their cytotoxic effects on CaCo2 cells using the DNA intercalating propidium iodide. The coexpressed Hbl tripartite toxin, the individual subunits and two coexpressed subunits were synthesized in the batch-mode. Subsequently the SN fraction ( a ) and the MF fraction ( b ) were analyzed. An NTC was used as a negative control. Standard deviations were calculated from triplicate samples of two independent experiments ( n = 6). ( c ) Morphological changes of CaCo2 cells exposed to the Hbl complex and an NTC volume equivalent.

Article Snippet: Therefore, the epithelial colorectal adenocarcinoma cell line CaCo2 (ATCC) was cultured in minimum essential medium (MEM) supplemented with 20% fetal calf serum (Merck, Biochrom GmbH, Berlin, Germany), 1% non-essential amino acids (Merck, Biochrom GmbH) and 1% penicillin/streptomycin (Merck, Biochrom GmbH).

Techniques: Membrane, Synthesized, Negative Control

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: Statins impair rotavirus replication. (a) Caco2 cells were infected with rotavirus SA11 strain (MOI 0.7) and subsequently treated with different concentrations of atorvastatin (n = 7), lovastatin (n = 7), or simvastatin (n = 5) for 48 hours, then the intra- (left) and extracellular (right) rotavirus RNA levels were measured by qRT-PCR, and (b) rotavirus VP4 protein were measured with 50 µM atorvastatin, lovastatin, and simvastatin treatment for 48 hours respectively by western blot. In MA104 cells, (c) treated as described in a, the intra- (left) and extracellular (right) rotavirus RNA levels with the three statins treatments respectively (n = 5), and (d) the expressions of the rotavirus VP4 protein, treated as described in b. In HSI organoids, (e) treated as described in a, the inner rotavirus RNA level with the treatments of the three statins respectively (n = 3), and (f) the expression of rotavirus VP4 protein, treated as described in b. (g) The titers of infectious rotavirus particles were measured by TCID 50 assays with 50 µM three statins treatments respectively in MA104 cells (n = 6). (h) The 96-hour time course experiments of atorvastatin (left), lovastatin (middle), and simvastatin (right) treatments respectively (n = 3) on the intracellular rotavirus RNA replication. All data presented as mean ± SEM, *p < .05, **p < .01, ***p < .001

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: Statins inhibit the expressions of rotavirus VP6 protein . (a) Schematic depiction of the inhibitors of cholesterol biosynthesis. Following the infection of rotavirus SA11 strain (MOI 0.7) with 50 µM atorvastatin, lovastatin, or simvastatin treatment for 48 hours respectively, the expression of rotavirus VP6 protein was observed by IF staining in Caco2 cells (b), MA104 cells (c) and HSI organoids (d). VP6 protein was green, and the nuclei of the cells were visualized by DAPI (blue). The results showed that statins significantly reduced the expression of rotavirus VP6 protein

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Expressing, Staining

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: shRNA-mediated HMGCR knockdown in Caco2 cells represses rotavirus replication . (a) Lentiviral shRNA vectors, targeting HMGCR gene or non-targeted control lentivirus were produced in HEK 293 T cells. Subsequently, the transductions of the lentiviral shRNA vectors were performed in Caco2 cells. qRT-PCR analysis was conducted to detect the RNA levels of HMGCR among shRNA 10951–10955 HMGCR vectors. (b) Based on the qRT-PCR analysis, the HMGCR knockdown effects of sh10952 and sh10955 vectors were confirmed by western blot analysis (n = 3). (c) The HMGCR knockdown significantly inhibited the intracellular rotavirus RNA replications post-infection 48 hours (n = 6), and (d) the decreased expressions of rotavirus VP4 protein by western blot analysis (n = 3). (e) The decreased expressions of rotavirus VP6 protein by HMGCR knockdown. VP6 protein was stained as green, and nuclei were visualized by DAPI (blue). All data presented as mean ± SEM, *p < .05, **p < .01, ***p < .001

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: shRNA, Knockdown, Control, Produced, Quantitative RT-PCR, Western Blot, Infection, Staining

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: The inhibitors of cholesterol biosynthesis impair rotavirus replication . With rotavirus SA11 strain (MOI 0.7) infection and subsequently treated with different concentrations of 6-fluoromevalonate for 48 hours. The intra- and extracellular rotavirus RNA levels were measured by qRT-PCR (n = 6) (left) and the expressions of rotavirus VP4 protein were tested by western blot (right) in Caco2 cells (a), and in MA104 cells (b). With ZA-A treatments, the intra- and extracellular rotavirus RNA levels (n = 9) (left) and the expressions of rotavirus VP4 protein (right) in Caco2 cells (c), and in MA104 cells (d). with 1.25 µM U18666A treatment, the intra- and extracellular rotavirus RNA levels (n = 6) (left) and the expressions of rotavirus VP4 protein (right) in Caco2 cells (e), and in MA104 cells (f). In HSI organoids, the inner rotavirus RNA level with 200 µM 6-fluoromevalonate (n = 5) (g), 50 µM ZA-A (n = 5) (h) and 1.25 µM U18666A (n = 4) (i); and the expressions of rotavirus VP4 protein with 200 µM 6-fluoromevalonate or 50 µM ZA-A treatment (left) (j), with 1.25 µM U18666A treatment (right) (k). All data presented as mean ± SEM, *p < .05, **p < .01, ***p < .001

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Quantitative RT-PCR, Western Blot

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: The rotavirus titers and VP6 protein expressions of 200 µM 6-Fluoromevalonate, 50 µM ZA-A or 1.25 µM U18666A treatment . The titers of infectious rotavirus particles were measured with 200 µM 6-Fluoromevalonate (a), 50 µM ZA-A (b) or 1.25 µM U18666A (c) treatment respectively (n = 6). Following the infection of rotavirus SA11 strain (MOI 0.7) with 200 µM 6-Fluoromevalonate or 50 µM ZA-A treatment respectively for 48 hours, the expression of rotavirus VP6 protein was observed in Caco2 cells (d), the expression of rotavirus VP6 protein was observed with 1.25 µM U18666A treatment in Caco2 cells (e), and the rotavirus VP6 expressions with the above treatments in MA104 cells (f) and HSI organoids (g). VP6 protein was green, and the nuclei of the cells were visualized by DAPI (blue). The results indicated that the inhibitors reduced the expressions of rotavirus VP6 protein

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Expressing

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: Enhancement of cholesterol production provokes rotavirus replication . With rotavirus SA11 strain (MOI 0.7) infection and subsequently treated with different concentrations of R-MA for 48 hours and subsequently the intra- and extracellular rotavirus RNA levels were measured by qRT-PCR (n = 5) (left) and the expressions of rotavirus VP4 protein were tested by western blot (right) in Caco2 cells (a), and in MA104 cells (b). With different concentrations of cholesterol treatment, the intra- and extracellular rotavirus levels (n = 5) (left) and the expressions of rotavirus VP4 protein (right) in Caco2 cells (c), and in MA104 cells (d). In HSI organoids, the inner rotavirus RNA level (n = 3) (left) and the expressions of rotavirus VP4 protein (right) with R-MA treatment (e), with cholesterol treatment (f). (g) The titers of infectious rotavirus particles were measured by TCID 50 assay upon R-MA (n = 6) (left) or cholesterol (n = 6) (right) treatment respectively in MA104 cells. (h) The time-course experiment of 150 µM cholesterol treatment on the intracellular rotavirus replication. All data presented as mean ± SEM, *p < .05, **p < .01, ***p < .001

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Quantitative RT-PCR, Western Blot

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: Rotavirus VP6 expressions by R-MA or cholesterol treatment . Rotavirus SA11 strain (MOI 0.7) infected Caco2 cells with 1,000 µM R-MA or 150 µM cholesterol treatment for 48 hours respectively in Caco2 cells (a), MA104 cells (b) and HSI organoids (c). Rotavirus VP6 protein was green, and the nuclei of the cells were visualized by DAPI (blue). The results showed that both of R-MA and cholesterol markedly increased the expressions of rotavirus VP6 protein

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: Infection

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: Cholesterol provokes rotavirus replication in the absence of HMGCR activity . With rotavirus SA11 strain (MOI 0.7) infection and subsequently treated with the combination of 50 µM atorvastatin and 150 µM cholesterol for 48 hours respectively. The intra- (left) and extracellular (right) rotavirus RNA levels were measured by qRT-PCR in Caco2 cells (n = 8) (a), and in MA104 cells (n = 6) (c). The expressions of rotavirus VP4 protein were measured by western blot in Caco2 cells (b), and in MA104 cells (d). With rotavirus SA11 infection (MOI 0.7), sh10952 and sh10955 HMGCR knockdown Caco2 cells were treated with 150 µM cholesterol for 48 hours. The intra- (left) and extracellular (right) rotavirus RNA levels (n = 4) (e) and the expression of rotavirus VP4 protein (f). All data presented as mean ± SEM, *p < .05, **p < .01, ***p < .001

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: Activity Assay, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Expressing

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: Rotavirus VP4 and VP6 proteins expressions of the combinations of atorvastatin or HMGCR knockdown and cholesterol treatment . Western blot analysis of the expressions of rotavirus VP4 protein with the combinations of 50 µM atorvastatin and 150 µM cholesterol in Caco2 cells (n = 3) (a), MA104 cells (n = 3) (b), and with the combinations of HMGCR knockdown and 150 µM cholesterol (n = 3) (c). The visual aspects of the combinations of 50 µM atorvastatin and 150 µM cholesterol in Caco2 cells (d), and MA104 cells (e), and with the combinations of HMGCR knockdown and 150 µM cholesterol treatment (f). The results showed that the combinations of atorvastatin or HMGCR knockdown and cholesterol treatment significantly increased the rotavirus replications

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: Knockdown, Western Blot

Journal: Gut Microbes

Article Title: Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

doi: 10.1080/19490976.2021.1955643

Figure Lengend Snippet: Canonical cholesterol biosynthesis is the rate-limiting step for a rotavirus clinical isolate 026 K replication in Caco2 cells . With the rotavirus 026 K strain infection, subsequently the intracellular rotavirus RNA levels were measured by qRT-PCR, after 48 hours treated with 50 µM atorvastatin (n = 4), or lovastatin (n = 4), or simvastatin (n = 6) treatment respectively (a), in sh10952 and sh10955 HMGCR knockdown Caco2 cells (b), with 6-fluoromevalonate treatment (n = 4) (c), with ZA-A treatment (n = 5) (d), with U18666A treatment (n = 6) (e), with R-MA treatment (n = 8) (f), with cholesterol treatment (n = 8) (g). All data presented as mean ± SEM, *p < .05, **p < .01, ***p < .001

Article Snippet: Human epithelial colorectal adenocarcinoma cell line Caco2, human embryonic kidney epithelial cell line 293 T (HEK 293 T) and African green monkey fetal kidney cell line MA104 were originally purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Quantitative RT-PCR, Knockdown