Journal: PLoS Pathogens

Article Title: Salmonella enterica serovar Typhimurium chitinases modulate the intestinal glycome and promote small intestinal invasion

doi: 10.1371/journal.ppat.1010167

Figure Lengend Snippet: (A) mRNA expression of S . Typhimurium chitinases during mid-log phase of growth in LB or DMEM/F12 + 10% FBS measured by RT-qPCR. Expression is normalized to the housekeeping gene gmk . n = 3. (B) Gentamicin protection assay of S . Typhimurium infected (MOI:1) colonic epithelial cells (T84). WT n = 14, Δ invA n = 14, Δ STM0233 n = 8, Δ chiA n = 8, Δ STM0233 Δ chiA n = 8. (C) Gentamicin protection assay of S . Typhimurium infected (MOI:1) small intestinal epithelial cells (IPEC-1). n = 6. (D) Adhesion assay of Cytochalasin D treated (2 μg/mL), S . Typhimurium infected (MOI:1) colonic epithelial cells (T84). WT n = 8, Δ invA n = 8, Δ STM0233 n = 8, Δ chiA n = 6, Δ STM0233 Δ chiA n = 8. (E) Adhesion assay of Cytochalasin D treated (2 μg/mL), S . Typhimurium infected (MOI:1) small intestinal epithelial cells (IPEC-1). n = 6. Bars represent mean ± SD. Statistics: (A) unpaired t -test. (B-E) Stars indicate significance compared to the WT control by (B, D) one-way ANOVA with Dunnett’s multiple comparison test or (C, E) mixed-effect analysis with Dunnett’s multiple comparison test. * = p<0.05, ** = p<0.01.

Article Snippet: The T84 colonic epithelial cell line (ATCC Cat# CCL-248; RRID:CVCL_0555) and IPEC-1 small intestinal epithelial cell line (DSMZ Cat# ACC-705, RRID:CVCL_2245) were grown in T75 flasks using 20 mL DMEM/F12 + 10% FBS + 1x antibiotic/antimycotic (Gibco).

Techniques: Expressing, Quantitative RT-PCR, Infection, Cell Adhesion Assay, Control, Comparison

Journal: International Journal of Oncology

Article Title: Butyrate supplementation regulates expression of chromosome segregation 1-like protein to reverse the genetic distortion caused by p53 mutations in colorectal cancer

doi: 10.3892/ijo.2022.5354

Figure Lengend Snippet: Different CSE1L expression levels in the colon cell lines. (A) Relative mRNA expression levels of CSE1L in CCD-18Co and FHC cells. (B) Relative mRNA expression levels of CSE1L in the CRC cell lines. (C) Relative mRNA expression levels of CSE1L in overconfluent Caco-2 cells. (D) Relative mRNA expression levels of c-Myc in overconfluent Caco-2 cells. (E) Protein expression level of CSE1L and c-Myc in overconfluent Caco-2 cells. (F) Relative mRNA expression levels of CSE1L in HCT116 cells with or without p53 expression. (G) Protein expression level of CSE1L in HCT116 cells with or without p53 expression. (H) Knockdown efficacy of p53 by mRNA level in HCT116 p53 +/+ cells. (I) Relative mRNA expression levels of CSE1L in HCT116 cells without or with p53 knockdown. (J) Protein expression levels of CSE1L in 5-FU-treated wild-type HCT116 cells. * P<0.05, ** P<0.01 and *** P<0.001. CRC, colorectal cancer; 5-FU, 5-fluouracil; shLuc, lentiviral construct targeting luciferase; shp53, lentiviral construct targeting p53; wt, wild-type; sh, short hairpin; CSE1L, chromo-some segregation 1-like protein.

Article Snippet: In addition, three human CRC cell lines (LS 174T, cat. no. CL-188; T84, cat. no. CCL-248; HCT116 p53 +/+ , cat. no. CCL-247; all from ATCC) expressing wild-type p53, two human CRC cell lines (SW480, cat. no. CCL-228; SW620, cat. no. CCL-227; both from ATCC) carrying the R273H and P309S double p53 mutation , in addition to two p53-null cell lines [Caco-2, cat. no. HTB-37, ATCC; HCT116 p53 −/− , a gift from Professor Bert Vogelstein (School of Medicine, Johns Hopkins University, Baltimore, USA)] ( , ), were used as tumorigenic cancer cells ( – ).

Techniques: Expressing, Knockdown, Construct, Luciferase

Journal: International Journal of Oncology

Article Title: Butyrate supplementation regulates expression of chromosome segregation 1-like protein to reverse the genetic distortion caused by p53 mutations in colorectal cancer

doi: 10.3892/ijo.2022.5354

Figure Lengend Snippet: Effect of butyrate supplementation and CSE1L overexpression on the genetic distortion caused by p53 mutations in colorectal cancer. Butyrate supplementation downregulates expression of CSE1L in p53-mutated CRC cells to mitigate the maligancy of CRC. CSE1L, chromosome segregation 1-like protein.

Article Snippet: In addition, three human CRC cell lines (LS 174T, cat. no. CL-188; T84, cat. no. CCL-248; HCT116 p53 +/+ , cat. no. CCL-247; all from ATCC) expressing wild-type p53, two human CRC cell lines (SW480, cat. no. CCL-228; SW620, cat. no. CCL-227; both from ATCC) carrying the R273H and P309S double p53 mutation , in addition to two p53-null cell lines [Caco-2, cat. no. HTB-37, ATCC; HCT116 p53 −/− , a gift from Professor Bert Vogelstein (School of Medicine, Johns Hopkins University, Baltimore, USA)] ( , ), were used as tumorigenic cancer cells ( – ).

Techniques: Over Expression, Expressing

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: CERS6‐AS1 is upregulated in colorectal cancer. (A) GEPIA ( http://gepia.cancer‐pku.cn/ ) prediction of the expression profile of CERS6‐AS1 in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). (B) The starBase database ( http://starbase.sysu.edu.cn/ ) shows the expression of CERS6‐AS1 in COAD. (C) CERS6‐AS1 expression in colorectal cancer cell lines (Caco‐2, HCT‐15, HCT‐15) and human normal colorectal mucosa epithelial cells (FHC). (D) A subcellular fractionation assay was used to detect the cellular location of CERS6‐AS1 in Caco‐2 and HCT‐15 cells. * p <0.05, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

Techniques: Expressing, Fractionation

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: CERS6‐AS1 deficiency suppresses malignant phenotypes of colorectal cancer cells. (A) The CERS6‐AS1 knockdown efficiency in colorectal cancer cells was measured by RT‐qPCR. (B,C) The viability and proliferation ability of Caco‐2 and HCT‐15 cells post‐transfection with sh‐CERS6‐AS1#1/#2 was assessed with CCK‐8 and colony formation assays. (D–F) The migration and invasion of Caco‐2 and HCT‐15 cells were assessed by Transwell assays. (G) The levels of EMT‐associated proteins (E‐cadherin, N‐cadherin, Slug, Twist) were measured using western blot after transfection of sh‐CERS6‐AS1#1/#2. (H) Cell stemness was measured using a sphere‐formation assay in cells after transfection with CERS6‐AS1#1/#2. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

Techniques: Knockdown, Quantitative RT-PCR, Transfection, CCK-8 Assay, Migration, Western Blot, Tube Formation Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: CERS6‐AS1 targets miR‐15b‐5p in colorectal cancer cells. (A) The miRNAs with binding sites for CERS6‐AS1 were searched in starBase. (B) The RNA pull‐down assay was used to explore the binding between the candidate miRNAs and CERS6‐AS1 in Caco‐2 and HCT‐15 cells. (C) The miR‐15b‐5p level in colon adenocarcinoma (COAD) was predicted by starBase. (D) MiR‐15b‐5p level in colorectal cancer cells. (E) The binding sequence of CERS6‐AS1 and miR‐15b‐5p was obtained from starBase. (F) The overexpression efficiency of miR‐15b‐5p in Caco‐2 and HCT‐15 cells using miR‐15b‐5p mimics. (G) A luciferase reporter assay was used to examine the binding between CERS6‐AS1 and miR‐15b‐5p after the indicated transfection. (H) RIP assay was used to detect whether CERS6‐AS1 and miR‐15b‐5p were enriched in the anti‐Ago2 precipitates. (I) The influence of CERS6‐AS1 silencing on miR‐15b‐5p expression was assessed using RT‐qPCR in Caco‐2 and HCT‐15 cells post‐transfection with CERS6‐AS1#1/#2. ** p <0.01, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

Techniques: Binding Assay, Pull Down Assay, Sequencing, Over Expression, Luciferase, Reporter Assay, Transfection, Expressing, Quantitative RT-PCR

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: SPTBN2 is directly targeted by miR‐15b‐5p. (A) The downstream target genes of miR‐15b‐5p were searched in starBase under the following screening conditions: CLIP‐data: strict stringency (≥5), degradome‐data: high stringency (≥3), pan‐cancer: 8 cancer types. (B) The expression of 10 candidate mRNAs in colorectal cancer cells posttransfection with miR‐15b‐5p mimics. (C) Survival analysis in patients with COAD with high or low levels of SPTBN2 based on starBase. (D,E) GEPIA showed SPTBN2 expression in COAD and READ, and starBase showed SPTBN2 expression in COAD. (F) SPTBN2 mRNA and protein levels in colorectal cancer cells were assessed using RT‐qPCR and western blot. (G) The potential binding sequences between SPTBN2 and miR‐15b‐5p from starBase. (H) SPTBN2 mRNA and protein expression was measured using RT‐qPCR and western blot in Caco‐2 and HCT‐15 cells transfected with sh‐CERS6‐AS1#1/#2. (I) The SPTBN2 mRNA level was assessed by RT‐qPCR in Caco‐2 and HCT‐15 cells transfected with miR‐15b‐5p mimics. (J) CERS6‐AS1 expression posttransfection with pcDNA3.1/CERS6‐AS1 in colorectal cancer cells. (K) A RIP assay was used to explore the relationship of CERS6‐AS1, miR‐15b‐5p and SPTBN2 in colorectal cancer cells. (L) A luciferase reporter assay was used to validate the interaction of CERS6‐AS1, miR‐15b‐5p, and SPTBN2 in colorectal cancer cells. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

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

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: CERS6‐AS1 promotes malignant behaviors in cells by upregulating SPTBN2 in colorectal cancer. (A) The SPTBN2 overexpression efficiency was examined using RT‐qPCR in colorectal cancer cells. (B,C) The viability and proliferation of Caco‐2 and HCT‐15 cells after transfection with sh‐CERS6‐AS1#2 or sh‐CERS6‐AS1#2+pcDNA3.1/SPTBN2 were measured by CCK‐8 and colony formation assays. (D–F) The migration and invasion capacities of Caco‐2 and HCT‐15 cells were assessed using Transwell assays in the sh‐CERS6‐AS1#2 and sh‐CERS6‐AS1#2+pcDNA3.1/SPTBN2 groups. (G) Western blot analysis showed the levels of proteins linked to the EMT process in Caco‐2 and HCT‐15 cells posttransfection with sh‐CERS6‐AS1#2 or sh‐CERS6‐AS1#2+pcDNA3.1/SPTBN2. (H) A sphere‐formation assay was used to examine cell stemness in the sh‐CERS6‐AS1#2 and sh‐CERS6‐AS1#2+pcDNA3.1/SPTBN2 groups. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

Techniques: Over Expression, Quantitative RT-PCR, Transfection, CCK-8 Assay, Migration, Western Blot, Tube Formation Assay

Journal: Cell host & microbe

Article Title: Systematic reconstruction of an effector-gene network reveals determinants of Salmonella cellular and tissue tropism.

doi: 10.1016/j.chom.2021.08.012

Figure Lengend Snippet: Figure 3. Identification of a minimal effector- gene network that supports intracellular Sal- monella replication and macrophage survival in vitro (A) Fold replication of the indicated STm strains in infected H1299, Hela, HCT116, and T84 cells. Sta- tistical significance was determined as in Figure 1B. (B–D) Confocal microscopy and TEM images of H1299 cells infected with the indicated GFP-ex- pressing STm strains for 18 h. The nucleus (blue) and endogenous Lamp1 (red) are shown. Arrowheads show the SCV membrane with multiple bacteria enclosed. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER LB broth Fisher Scientific Cat#BP9722 LB agar Fisher Scientific Cat#BP9724 Nutrient Broth Becton Dickinson Cat#234000 Paraformaldehyde (4%) Alfa Aesar Cat#47392 PBS Sigma Cat#D8537 RPMI Medium 1640 Thermo Fisher Cat#11875 Saponin MP Biomedicals Cat#102855 Sodium Chloride Fisher Scientific Cat#BP358 Streptomycin sulfate Fisher Scientific Cat#BP910 Sucrose Fisher Science Education Cat#S25590 Triton X-100 Fisher Scientific Cat#BP151 Tryptone Becton Dickinson Cat#211705 Yeast Extract Becton Dickinson Cat#212750 Experimental models: Cell lines Hela ATCC CCL-2 RAW264.7 ATCC TIB-71 HCT116 Mendel Lab N/A T84 Winter Lab N/A H1299 Minna Lab N/A Experimental models: Organisms/strains C57BL/6J mice Jackson Lab 000664 129X1/SvJ mice Jackson Lab 000691 C57BL/6J Nramp1G169 mice (Arpaia et al., 2011) N/A Oligonucleotides See Tables S1–S3 for full list of primers used in this study This Study See Tables S1–S3 Recombinant DNA GFP expression plasmid (Murphy et al., 2002) pBBR1MCS-6y mCherry expression plasmid (Burnaevskiy et al., 2013) pDP151 ori(R6K) mobRP4 Cmr Tetr sacRB (Kingsley et al., 1999) pRDH10 Template plasmid for FRT-flanked KanR cassette (Datsenko and Wanner, 2000) pKD4 Red recombinase expression plasmid (Datsenko and Wanner, 2000) pKD46 Helper plasmid to eliminate the KanR cassette (Datsenko and Wanner, 2000) pCP20 downstream region of STm spvR in pRDH10 This Study pNA101 downstream region of STm pipAB in pRDH10 This Study pNA102 downstream region of STm pipB2 in pRDH10 This Study pNA103 downstream region of STm gtgA in pRDH10 This Study pNA104 downstream region of STm sifB in pRDH10 This Study pNA105 downstream region of STm gtgEsseI in pRDH10 This Study pNA106 downstream region of STm steBsseJ in pRDH10 This Study pNA107 downstream region of STm pipD in pRDH10 This Study pNA108 downstream region of STm sseL in pRDH10 This Study pNA109 downstream region of STm gogBsteE in pRDH10 This Study pNA110 downstream region of STm sopD2 in pRDH10 This Study pNA111 downstream region of STm slrp in pRDH10 This Study pNA112 downstream region of STm steA in pRDH10 This Study pNA113 downstream region of STm steD in pRDH10 This Study pNA114 downstream region of STm sspH2 in pRDH10 This Study pNA115 downstream region of STm sseK2 in pRDH10 This Study pNA116 (Continued on next page) e4 Cell Host & Microbe 29, 1531–1544.e1–e9, October 13, 2021

Techniques: In Vitro, Infection, Confocal Microscopy, Membrane, Bacteria