ls174t colorectal adenocarcinoma cells  (ATCC)

Journal: bioRxiv

Article Title: TTC7A organizes glandular lumen formation in the intestine through a Class II phosphatidylinositol 3-kinase

doi: 10.1101/2025.03.22.644724

Figure Lengend Snippet: a. Representative confocal images with inverted micrographs showing immunofluorescent staining of actin caps (arrowheads) in LS174T:W4 control (WT) TTC7A-knockdown (KD) cells; actin (green) and villin (red), indicated by arrowheads. b. Quantification of secondary actin cap formation in TTC7A-KD cells. Mean of 3 experiments with 5 images per condition. (*p=<0.002). c. Quantification of actin cap formation across polarization in W4 cells; data represent two technical replicates, (n=5 images/time point). d. Representative confocal imaging with inverted micrographs showing endogenous localization of TTC7A (red) and PI4KIIIΑ (green) during W4 cell polarization; actin caps marked by red arrows. e. Representative plot profiles showing normalized intensity vs. cell length for the localization of Actin (teal) with DAPI (blue), TTC7A (red), and PI4KIIIΑ (green) at the endpoint of polarization. f. Representative confocal imaging of control colonoids showing endogenous TC7A (red) and PI4KIIIΑ (green) subcellular localization in early lumens and in mature lumens. Dashed boxes indicate the zoomed insets of the lumen. Insets show diverging TTC7A and PI4KIIIΑ in early lumen formation, and colocalization of TTC7A-PI4KIIIΑ in colonoids with a mature lumen by orange arrows. Grey-scale images are inverted black and white confocal images. g. Pearson’s correlation of TTC7A-PI4KIIIΑ colocalization in control vs. patient-derived colonoids. Data is representative of 4-6 experiments, One-way ANOVA, Tukey’s posthoc testing. ****<0.0001. h. Representative western blot on CO-IP TTC7A-pulldown probing for PI4KIIIΑ (top) in early lumen formation in three control and three patient colonoid lines. Data is representative of 6 experimental replicates. Western blot on whole cell lysates in three control and three patient colonoid lines probing for PI4KIIIΑ, TTC7A and actin. Data is representative of >6 experimental replicates.

Article Snippet: Constructs for PI4KIIIΑ (ThermoFisher, AM51331), PIK3C2A (ThermoFisher, 4390824, Assay ID 10508), and Efr3a (ThermoFisher, AM16074, ID 219474) were purchased from ThermoFisher. siRNA constructs were transiently transfected into LS174T:W4 cells using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher 13778030) according to the manufacturer’s protocol, 24 hours prior to the induction of polarization.

Techniques: Staining, Control, Knockdown, Imaging, Derivative Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: TTC7A organizes glandular lumen formation in the intestine through a Class II phosphatidylinositol 3-kinase

doi: 10.1101/2025.03.22.644724

Figure Lengend Snippet: a. Representative confocal images with inverted micrographs showing immunofluorescent staining of actin caps (arrowheads) in LS174T:W4 control (WT) TTC7A-knockdown (KD) cells; actin (green) and villin (red), indicated by arrowheads. b. Quantification of secondary actin cap formation in TTC7A-KD cells. Mean of 3 experiments with 5 images per condition. (*p=<0.002). c. Quantification of actin cap formation across polarization in W4 cells; data represent two technical replicates, (n=5 images/time point). d. Representative confocal imaging with inverted micrographs showing endogenous localization of TTC7A (red) and PI4KIIIΑ (green) during W4 cell polarization; actin caps marked by red arrows. e. Representative plot profiles showing normalized intensity vs. cell length for the localization of Actin (teal) with DAPI (blue), TTC7A (red), and PI4KIIIΑ (green) at the endpoint of polarization. f. Representative confocal imaging of control colonoids showing endogenous TC7A (red) and PI4KIIIΑ (green) subcellular localization in early lumens and in mature lumens. Dashed boxes indicate the zoomed insets of the lumen. Insets show diverging TTC7A and PI4KIIIΑ in early lumen formation, and colocalization of TTC7A-PI4KIIIΑ in colonoids with a mature lumen by orange arrows. Grey-scale images are inverted black and white confocal images. g. Pearson’s correlation of TTC7A-PI4KIIIΑ colocalization in control vs. patient-derived colonoids. Data is representative of 4-6 experiments, One-way ANOVA, Tukey’s posthoc testing. ****<0.0001. h. Representative western blot on CO-IP TTC7A-pulldown probing for PI4KIIIΑ (top) in early lumen formation in three control and three patient colonoid lines. Data is representative of 6 experimental replicates. Western blot on whole cell lysates in three control and three patient colonoid lines probing for PI4KIIIΑ, TTC7A and actin. Data is representative of >6 experimental replicates.

Article Snippet: LS174T-W4 cells were seeded on a 24-well glass bottom plate (Cellvis: P24-1.5H-N) with a density of 0.1 x 10 per well.

Techniques: Staining, Control, Knockdown, Imaging, Derivative Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: TTC7A organizes glandular lumen formation in the intestine through a Class II phosphatidylinositol 3-kinase

doi: 10.1101/2025.03.22.644724

Figure Lengend Snippet: a. Representative confocal images with inverted micrographs showing immunofluorescent staining of actin caps (arrowheads) in LS174T:W4 control (WT) TTC7A-knockdown (KD) cells; actin (green) and villin (red), indicated by arrowheads. b. Quantification of secondary actin cap formation in TTC7A-KD cells. Mean of 3 experiments with 5 images per condition. (*p=<0.002). c. Quantification of actin cap formation across polarization in W4 cells; data represent two technical replicates, (n=5 images/time point). d. Representative confocal imaging with inverted micrographs showing endogenous localization of TTC7A (red) and PI4KIIIΑ (green) during W4 cell polarization; actin caps marked by red arrows. e. Representative plot profiles showing normalized intensity vs. cell length for the localization of Actin (teal) with DAPI (blue), TTC7A (red), and PI4KIIIΑ (green) at the endpoint of polarization. f. Representative confocal imaging of control colonoids showing endogenous TC7A (red) and PI4KIIIΑ (green) subcellular localization in early lumens and in mature lumens. Dashed boxes indicate the zoomed insets of the lumen. Insets show diverging TTC7A and PI4KIIIΑ in early lumen formation, and colocalization of TTC7A-PI4KIIIΑ in colonoids with a mature lumen by orange arrows. Grey-scale images are inverted black and white confocal images. g. Pearson’s correlation of TTC7A-PI4KIIIΑ colocalization in control vs. patient-derived colonoids. Data is representative of 4-6 experiments, One-way ANOVA, Tukey’s posthoc testing. ****<0.0001. h. Representative western blot on CO-IP TTC7A-pulldown probing for PI4KIIIΑ (top) in early lumen formation in three control and three patient colonoid lines. Data is representative of 6 experimental replicates. Western blot on whole cell lysates in three control and three patient colonoid lines probing for PI4KIIIΑ, TTC7A and actin. Data is representative of >6 experimental replicates.

Article Snippet: Caco2 and LS174T:W4 cells were transduced with TTC7A-lenti particles using 10ug/ml Polybrene (Santa Cruz Biotechnology, sc-134220).

Techniques: Staining, Control, Knockdown, Imaging, Derivative Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: Frontiers in Microbiology

Article Title: Clostridium butyricum ameliorates indomethacin-induced enteropathy by promoting MUC2 secretion via suppressing the Notch pathway

doi: 10.3389/fmicb.2025.1509876

Figure Lengend Snippet: Clostridium butyricum supernatant (CBS) promoted the expression of MUC2 via suppression of Notch signaling pathway in LS174T cells. (A) Treatment with different concentrations of CBS (1, 2, 5, 7, and 10%) for 48 h. Cell viability was measured by CCK-8 ( n = 6). (B,C) Relative expression of MATH-1, HES-1, Notch-1, and MUC2 mRNA induced by different concentrations of CBS ( n = 4). (D,E) Western blotting and quantitative analysis of MATH-1, HES-1, Notch-1, and MUC2 proteins induced by different concentrations of CBS ( n = 3). (F,G) Relative expression of MATH-1, HES-1, Notch-1, and MUC2 mRNA induced by CBS and different concentrations of valproic acid (VPA), an agonist of Notch signaling ( n = 4). (H,I) Western blotting and quantitative analysis of MATH-1, HES-1, Notch-1, and MUC2 proteins induced by CBS and different concentrations of VPA ( n = 3). Data were presented as mean ± standard deviation. * p < 0.05 and ** p < 0.01.

Article Snippet: The LS174T cells were cultured in Dulbecco’s MEM (Solarbio, China) with 15% FBS, 0.5% penicillin–streptomycin, and 0.5% non-essential amino acids.

Techniques: Expressing, CCK-8 Assay, Western Blot, Standard Deviation