Journal: Nature communications

Article Title: Multiple structures of RNA polymerase II isolated from human nuclei by ChIP-CryoEM analysis.

doi: 10.1038/s41467-025-59580-x

Figure Lengend Snippet: Fig. 2 | Details of the EC-SPT4/5-ELOF1-SPT6 structure. a Domain architectures of the elongation factors. The known domains are indicated. b Close-up view of ELOF1 (purple). c Close-up view around the upstream DNA. d Close-up view of the SPT5 KOW2, KOW3, KOWx, and KOW4 domains. Each KOW domain is indicated with dotted circles. SPT6 is omitted for clarity. e Close-up view of the KOW5 domain. f The SPT6-SPT5 KOW3-RPB4/7 stalk interaction. g The SPT5 KOW1-SPT6

Article Snippet: Themembraneswerewashedwith TBS-T (20mMTris-HCl (pH 7.5), 137mM NaCl, and 0.1% Tween-20) three times and then incubated overnight with anti-FLAG M2 antibody (Sigma # F3165, 1:3000), anti-SPT5 antibody (Proteintech#16511-1-AP, 1:1000), antiSPT6 antibody (Cell Signaling #15616, 1:2000), anti-PAF1 antibody (Cell Signaling #12883, 1:300), anti-RTF1 antibody (Proteintech #12170-1-AP, 1:1000), anti-MED17 antibody (Proteintech#11505-1-AP, 1:2000), or anti-XPB antibody (Cell Signaling #8746, 1:300) in Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO #NKB-101).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: mRNA levels of CrT1 along the length of mouse ( A ) and rat ( B ) intestinal mucosa. Total RNA isolated from scrapped mucosa was amplified with species (mouse or rat)-specific gene ( CrT1 ) primers for real-time PCR quantification. Data represent the relative expression of CrT1 normalized to respective GAPDH mRNA (internal control) in different segments of the intestine (Jej: jejunum; Ile: ileum; PC: proximal colon; DC: distal colon). Data represent mean ± SEM. * p < 0.05, **** p < 0.0001 and ns = not significant between groups as indicated.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques: Isolation, Amplification, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: Protein levels of CrT1 along the length of mouse ( A ), rat ( B ), and human ( C ) intestinal mucosa. Tissue lysates prepared from scrapped mucosa were subjected to 10% SDS-polyacrylamide gel, transferred to PVDF membrane, and probed with anti-CrT1 antibody. The upper panels are representative blots showing the relative expression of CrT1 in different regions of the intestine with β-actin as the loading control. For mice samples, 75 µg protein was loaded per well of the gel, and for rats and human samples, 50 µg was loaded. Lower panels show the results of densitometric analysis of band intensities of CrT1 normalized to that of β-actin. Data represent mean ± SEM. *** p < 0.001, **** p < 0.0001 and ns = not significant between groups as indicated.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques: Membrane, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: CrT1 is localized to the luminal (brush border) membrane in rat jejunum. OCT sections of jejunum were immunostained for CrT1 (red), the apical membrane marker villin (green), and nuclei (blue) as described in Methods. Uniform expression of CrT1 in cells along the length of the villi is shown in red. BBM localization of villin in cells along the length of the villi is shown in green. Colocalization of CrT1 with villin in the BBM of villus cells is shown in the merged image. A representative image is shown. Scale bar—100 µm.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques: Membrane, Marker, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: Proinflammatory cytokines inhibit CrT1 function (Na + -dependent 14 C-creatine uptake) in Caco-2 ( A ) and IEC-6 ( B ) cells. Post-confluent cell monolayers grown on 12-well transwells were treated with TNFα or IL1β (10 ng/mL) from the basolateral compartment for 24 h. Na + -dependent uptake of 14 C-creatine was determined in the presence or absence of the competitive CrT1 substrate β-guanidinopropionic acid (GPA, 1 mM). Results are expressed as nmoles-mg protein −1 × 30 min −1 . Data represent mean ± SEM. **** p < 0.0001 between groups as indicated.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: Proinflammatory cytokines decrease mRNA and protein levels of CrT1 in IECs. Caco-2 and IEC-6 cells grown on transwell inserts were treated with TNFα or IL1β (10 ng/mL) from the basolateral compartments for 24 h. mRNA levels of CrT1 in total RNA from Caco-2 ( A ) and IEC-6 ( B ) cells were amplified by real-time QRT-PCR using species-specific (human or rat) primers for the CrT1 gene. Similarly, protein levels of CrT1 in the total lysates of Caco-2 (( C ), upper panel ) and IEC-6 (( D ), upper panel ) were determined by Western blot. Lower panels show the respective densitometric analysis of the band intensities of CrT1 normalized to that of β-actin. Data represent mean ± SEM. *** p < 0.001 and **** p < 0.0001 between groups as indicated.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques: Amplification, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: TNFα and IL1β treatments decrease CrT1 promoter activity in Caco-2 cells. A 1029 bp fragment of the human CrT1 promoter region was transfected into Caco-2 cells using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). Twenty-four hours post-transfection, cells were treated with TNFα or IL1β (10 ng/mL) for 24 h. Promoter activity (luciferase units) was determined as described in the Methods section. Data represent mean ± SEM. ** p < 0.01 and *** p < 0.001 between groups as indicated.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques: Activity Assay, Transfection, Luciferase

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: CrT1 expression is decreased in vivo in SAMP1 small intestinal mucosa compared to AKR mice. ( A ) CrT1 mRNA levels in total RNA extracted from SAMP1/AKR small intestinal mucosa were measured by QRT-PCR, utilizing gene-specific mouse primers. Data represent the relative expression of CrT1 normalized to respective GAPDH mRNA (internal control). ( B ) CrT1 protein levels in total lysate extracted from SAMP1/AKR small intestinal mucosa were measured by Western blot. The upper panel shows a representative blot. The lower panel shows the densitometric analysis of the band intensities of CrT1 normalized to that of β-actin. Data represent mean ± SEM. **** p < 0.0001 between groups as indicated.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques: Expressing, In Vivo, Quantitative RT-PCR, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: CrT1 expression is decreased in SAMP1 small intestinal organoids compared to AKR organoids. ( A ) CrT1 mRNA levels in total RNA extracted from SAMP1/AKR small intestinal organoids were measured by QRT-PCR, utilizing gene-specific mouse primers. Data represent the relative expression of CrT1 mRNA normalized to respective β2-microglobulin (B2M) mRNA (internal control). ( B ) CrT1 protein levels in total lysates made from SAMP1/AKR small intestinal organoids were measured by Western blot. The upper panel shows a representative blot. The lower panel shows the densitometric analysis of the band intensities of CrT1 normalized to that of β-actin. Data represent mean ± SEM. **** p < 0.0001 between groups as indicated.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

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

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: AIEC infection decreases CrT1 expression in vitro. Cell monolayers (Caco-2 or IEC-6) were treated from the apical surface for 8 h with a 1:10 diluted bacterial suspension of an exponentially grown overnight culture of AIEC LF-82 showing an absorbance (600 nm) = 0.2. ( A ) CrT1 mRNA levels in total RNA extracted from Caco-2/IEC-6 cells were measured by QRT-PCR, utilizing gene-specific human/rat primers. Data represent the relative expression of CrT1 mRNA normalized to respective GAPDH mRNA (internal control). ( B ) CrT1 protein levels in total lysates of Caco-2/IEC-6 cells were measured by Western blot. The upper panel shows a representative blot. The lower panel shows the densitometric analysis of the band intensities of CrT1 normalized to that of β-actin. Data represent mean ± SEM. *** p < 0.001 between groups as indicated.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques: Infection, Expressing, In Vitro, Suspension, Quantitative RT-PCR, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: AIEC infection diminishes CrT1 in SAMP1 organoid-derived monolayers with no significant effects on AKR organoid monolayers. Two-dimensional monolayers of crypt-derived small intestinal organoids generated from SAMP1/AKR mice were treated from the apical surface for 8 h with a 1:10 diluted bacterial suspension of an exponentially grown overnight culture of AIEC LF-82 showing an absorbance (600 nm) = 0.2. (A) CrT1 mRNA levels in total RNA extracted from SAMP1/AKR organoid monolayers (ORG-ML) were measured by QRT-PCR, utilizing gene-specific mouse primers. Data represent the relative expression of CrT1 mRNA normalized to respective β2-microglobulin (B2M) mRNA (internal control). Data represent mean ± SEM. *** p < 0.001 and ns = not significant between groups as indicated.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques: Infection, Derivative Assay, Generated, Suspension, Quantitative RT-PCR, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection

doi: 10.3390/ijms25126537

Figure Lengend Snippet: Schematic representation of the potential cause-and-effect relationship between CrT1 deficiency and IBD-associated chronic mucosal inflammation. CrT1 plays a critical role in maintaining energy homeostasis in intestinal epithelial cells (IECs) via mediating the absorption of dietary creatine (Cr), a potent energy nutrient. Optimal Cr availability ensures proper functioning of the creatine–phosphocreatine (Cr-pCr) shuttle that supplies energy to IECs. In inflammatory bowel disease (IBD), elevated levels of proinflammatory cytokines, such as TNFα and IL1β, diminish CrT1 expression and function. Further, IBD-associated dysbiosis triggers colonization of the inflamed mucosa by several opportunistic pathogens, adherent invasive E. coli (AIEC) being a predominant one. AIEC infection further downregulates CrT1 which results in Cr deficiency and impaired Cr-pCr shuttle. Thus, IECs become energy deficient at a time of high energy demand to combat the effects of active inflammation and dysbiosis. Therefore, impaired Cr transport into IECs could be a major contributing factor in sustaining chronic inflammation in IBD as well as for its relapsing and recurring episodic nature.

Article Snippet: Immunoblotting was carried out with anti-CrT1 antibody (Proteintech, Rosemont, IL, USA, Catalog #20299-1-AP, dilution 1:1000) and with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog #sc47778, dilution 1:2000) as the loading control.

Techniques: Expressing, Infection