Journal: Journal of Biomedical Science

Article Title: Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells

doi: 10.1186/s12929-022-00814-1

Figure Lengend Snippet: ChREBP expression is reduced in the renal cortex of db/db mice treated with the LPAR inhibitor, ki16425. Eight-week-old wild-type and db/db mice were intraperitoneally injected with the vehicle or ki16425 (10 mg/kg) for 8 weeks on a daily basis, following which the mice were sacrificed. A The level of ChREBP protein in the renal cortex was analyzed by western blotting, quantified using ImageJ software, and normalized to those of β-actin. B The level of ChREBP mRNA in the renal cortex was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The data are presented as the mean ± SEM (n = 3/group); *p < 0.05, **p < 0.01. C Representative images depicting the colocalization of α-SMA (green) and ChREBP (red) in the kidney tissue sections of mice. The nuclei were counterstained with DAPI (blue). The white arrows indicate the cytosolic expression of ChREBP, and the yellow arrows indicate the nuclear expression of ChREBP in α-SMA-positive cells. Scale bars, 20 μm; n = 3

Article Snippet: For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions.

Techniques: Expressing, Injection, Western Blot, Software, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Journal of Biomedical Science

Article Title: Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells

doi: 10.1186/s12929-022-00814-1

Figure Lengend Snippet: LPA induces the expression of ChREBP and fibrotic factors in SV40 MES13 cells. A SV40 MES13 cells were treated with 10 µM LPA for the indicated durations. The level of ChREBP protein was analyzed by western blotting, quantified using ImageJ software, and normalized to that of β-actin. The data are presented as the mean ± SEM of results obtained from three independent experiments. *p < 0.05 vs. control. B SV40 MES13 cells were seeded in 4-well chambers and treated with LPA in the presence or absence of ki16425 for 3 h. Immunocytochemistry analysis was performed using an anti-ChREBP antibody and an Alexa Fluor 546-conjugated secondary antibody. The nuclei were counterstained with DAPI (blue) (original magnification, 400 ×; the scale bars represent 20 μm). C The SV40 MES13 cells were treated with LPA in the presence or absence of ki16425 for 3 h. The protein levels of ChREBP, fibronectin, TGF-β, and IL-1β were analyzed by western blotting, quantified using ImageJ software, and normalized to those of β-actin. The data are presented as the mean ± SEM of results obtained from three independent experiments. *p < 0.05 vs. control (Con); # p < 0.05 vs. LPA

Article Snippet: For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Software, Control, Immunocytochemistry

Journal: Journal of Biomedical Science

Article Title: Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells

doi: 10.1186/s12929-022-00814-1

Figure Lengend Snippet: ChREBP regulates the LPA-induced expression of fibrotic factors in SV40 MES13 cells. A and B SV40 MES13 cells were transfected with a control siRNA (siCon) or ChREBP siRNA (siChREBP), and treated with 10 µM LPA for 3 h. The mRNA (A) and protein ( B ) levels of ChREBP, fibronectin, TGF-β, and IL-1β were analyzed by qRT-PCR (n = 5) and western blotting (n = 3), respectively. C and D The SV40 MES13 cells were co-transfected with vectors expressing ChREBP and Mlx (ChREBP/Mlx), or transfected with an empty pcDNA vector (Con), and treated with 10 µM LPA for 3 h. The mRNA ( C ) and protein ( D ) levels of ChREBP, fibronectin, TGF-β, and IL-1β were analyzed by qRT-PCR (n = 5) and western blotting (n = 3), respectively. The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005 vs. siCon-BSA or Con-BSA; # p < 0.05, ## p < 0.01, ### p < 0.005 vs. siCon-LPA or Con-LPA

Article Snippet: For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Western Blot, Plasmid Preparation

Journal: Journal of Biomedical Science

Article Title: Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells

doi: 10.1186/s12929-022-00814-1

Figure Lengend Snippet: LPA induces the production of ROS, and ROS scavenging decreases the LPA- or ROS-induced expression of ChREBP and fibrotic factors in SV40 MES13 cells. A SV40 MES13 cells were treated with 10 µM LPA for the indicated durations. B SV40 MES13 cells were treated with LPA in the presence or absence of ki16425 for 1 h. A and B The intracellular levels of ROS were measured by flow cytometry with DCFH-DA. The values in the representative flow cytometry histograms indicate the fluorescence intensity of DCF in the whole cells. The relative fluorescence intensity levels of DCF were compared with the production of ROS in the control cells. C The cells were pretreated with NAC for 1 h and subsequently treated with LPA for 3 h. D The cells were pretreated with NAC for 1 h and subsequently treated with H 2 O 2 for 1 h. C and D The protein levels of ChREBP, fibronectin, TGF-β, and IL-1β were analyzed by western blotting, quantified using ImageJ software, and normalized to those of β-actin. The data are presented as the mean ± SEM of results obtained from three independent experiments. *p < 0.05, **p < 0.01 vs. Con; # p < 0.05, ## p < 0.01 vs. LPA or H 2 O 2

Article Snippet: For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Fluorescence, Control, Western Blot, Software

Journal: Journal of Biomedical Science

Article Title: Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells

doi: 10.1186/s12929-022-00814-1

Figure Lengend Snippet: LPA induces the expression of ChREBP and fibrotic factors via Smurf2 in SV40 MES13 cells. A SV40 MES13 cells were treated with LPA, either in the presence or absence of ki16425, or treated with Heclin (Hec) for 3 h. B and C The SV40 MES13 cells were transfected with a control siRNA (siCon) or Smurf2 siRNA (siSmurf2, siSm2) for 6 h, then the medium was replaced with SFM for 16–18 h, and treated with LPA for 3 h. The proteins levels of ChREBP, Smurf2, fibronectin, TGF-β, and IL-1β were analyzed by western blotting, quantified using ImageJ software, and normalized to those of β-actin. The data are presented as the mean ± SEM of results obtained from three to five independent experiments. *p < 0.05, **p < 0.01 vs. Con or siCon-BSA; # p < 0.05, ## p < 0.01, ### p < 0.005 vs. LPA or siCon-LPA

Article Snippet: For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Control, Western Blot, Software

Journal: Journal of Biomedical Science

Article Title: Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells

doi: 10.1186/s12929-022-00814-1

Figure Lengend Snippet: LPA inhibits the ubiquitination of ChREBP by downregulating Smurf2 in SV40 MES13 cells. A SV40 MES13 cells were treated with LPA in the presence or absence of ki16425 for 3 h. B The cells were transfected with a control siRNA (siCon) or Smurf2 siRNA (siSmurf2) for 6 h, then the medium was replaced with SFM for 16–18 h, and subsequently treated with LPA for 3 h. C The cells were transfected with pCMV5B-Flag-Smurf2 wt (Smurf2 wt) or pCMV5B-Flag-Smurf2 C716A (Smurf2 C716A) for 6 h, then the medium was replaced with SFM for 16–18 h, and subsequently treated with LPA for 3 h. (A-C) The cell lysates were subjected to immunoprecipitation studies by incubating with an anti-ChREBP antibody followed by blotting with anti-ubiquitin antibody. The protein levels of ubiquitinated-ChREBP (Ub-ChREBP), ChREBP, and Smurf2 were analyzed by western blotting. Representative images of the blots from three independent experiments are depicted, where the red brackets indicate Ub-ChREBP

Article Snippet: For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions.

Techniques: Ubiquitin Proteomics, Transfection, Control, Immunoprecipitation, Western Blot

Journal: Journal of Biomedical Science

Article Title: Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells

doi: 10.1186/s12929-022-00814-1

Figure Lengend Snippet: Inhibition of Akt signaling suppresses the alterations in the expression of ChREBP and Smurf2 induced by LPA. A SV40 MES13 cells were pretreated with A6730 (Akt1/2 kinase inhibitor, 4 μM) or Ly 294002 (PI3K inhibitor, 10 μM) for 1 h, and subsequently treated with LPA and/or ki16425 for 3 h. B SV40 MES13 cells were pretreated with NAC for 1 h and subsequently treated with LPA for 3 h. A and B The protein levels of p-Akt, ChREBP, and Smurf2 were analyzed by western blotting, quantified using ImageJ software, and normalized to those of β-actin. The data are presented as the mean ± SEM of results obtained from four independent experiments. *p < 0.05, **p < 0.01 vs. vehicle only (control); # p < 0.05, ## p < 0.01 vs. LPA only

Article Snippet: For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions.

Techniques: Inhibition, Expressing, Western Blot, Software, Control

Journal: Journal of Biomedical Science

Article Title: Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells

doi: 10.1186/s12929-022-00814-1

Figure Lengend Snippet: LPA increases Traf4-mediated ubiquitination of Smurf2 in SV40 MES13 cells. A SV40 MES13 cells were pretreated with NAC for 1 h and subsequently treated with LPA for 3 h. The level of Smurf2 protein was analyzed by western blotting, quantified using ImageJ software, and normalized to that of β-actin. B and C SV40 MES13 cells were treated with 10 µM LPA for the indicated durations. The mRNA levels of Smurf2 ( B ) and ubiquitin ligases ( C ), including Traf4, Trib3, Ttc3, and USP11, were determined by qRT-PCR. D – F SV40 MES13 cells were treated with LPA in the presence or absence of ki16425 for 3 h. D The mRNA levels of Traf4 and Trib3 were determined by qRT-PCR. E The level of Traf4 protein was analyzed by western blotting, quantified using ImageJ software, and normalized to that of β-actin. F The cell lysates were subjected to immunoprecipitation studies by incubating with an anti-Smurf2 antibody followed by blotting with anti-ubiquitin antibody. The protein levels of ubiquitinated-Smurf2 (Ub-Smurf2), ChREBP, and Smurf2 were analyzed by western blotting. Representative images of the blots from three independent experiments are depicted, where the red brackets indicate Ub-Smurf2. G SV40 MES13 cells were transfected with a control (siCon) or Traf4 (siTraf4) siRNA for 6 h, then the medium was replaced with SFM for 16–18 h, and treated with LPA for 3 h. The proteins levels of Traf4 and p-Akt were analyzed by western blotting, quantified using ImageJ software, and normalized to those of β-actin and Akt, respectively. The data are presented as the mean ± SEM of results obtained from four independent experiments. *p < 0.05, **p < 0.01 vs. vehicle only (control) or siCon-BSA; # p < 0.05, ## p < 0.01 vs. LPA only or siCon-LPA

Article Snippet: For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions.

Techniques: Ubiquitin Proteomics, Western Blot, Software, Quantitative RT-PCR, Immunoprecipitation, Transfection, Control

Journal: Journal of Biomedical Science

Article Title: Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells

doi: 10.1186/s12929-022-00814-1

Figure Lengend Snippet: Schematic representation of the mechanism by which LPA induces ChREBP-mediated fibrosis, via the ROS/Akt-dependent downregulation of Smurf2 in renal mesangial cells. Created with BioRender.com. Red and blue arrows indicate upregulated and downregulated responses, respectively

Article Snippet: For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions.

Techniques:

Journal: Cells

Article Title: Glucose-Sensing Carbohydrate Response Element-Binding Protein in the Pathogenesis of Diabetic Retinopathy.

doi: 10.3390/cells14020107

Figure Lengend Snippet: Figure 1. The immunohistochemical analysis of human control and diabetic retinas. (A) The retinas were subjected to treatment with anti-ChREBP antibody. The ChREBP immunoreactivity in human control (A,C) and diabetic (B,D) retinas. In the diabetic retina, robust staining of ChREBP (green) was detected in the photoreceptors, the cells of the INL (E), endothelial cells (F,H), retinal pigment epithelial cells (G), and retinal ganglion cells (I). The co-localization of ChREBP in the nuclei (red) of retinal cells is indicated in yellow. (B) The MondoA immunoreactivity in human control (A) and diabetic (B) retinas is shown in green. In the diabetic retina, robust staining was detected in the cones (C) and the cells of the INL (C). The localization of MondoA in the nuclei is shown in yellow (D).

Article Snippet: With lentiviral particles expressing ChREBP (Cat#: RC220626L2V OriGene Co., Rockville, MD, USA) was conducted using 100 μl of suspension.

Techniques: Immunohistochemical staining, Control, Staining

Journal: Cells

Article Title: Glucose-Sensing Carbohydrate Response Element-Binding Protein in the Pathogenesis of Diabetic Retinopathy.

doi: 10.3390/cells14020107

Figure Lengend Snippet: Figure 2. Expression of Mondo family proteins in hyperglycemic mouse retinas. (A) The RNAscope technique revealed enhanced ChREBP mRNA expression (green) in 12-week-old db/db retinas. The retinal ganglion cells (RGCs), Müller cells (highlighted in right inserts), and photoreceptors are responsive to hyperglycemia, showing increased ChREBP expression. (B) Mouse retinal explants were cultured in a medium supplemented with either high glucose or an equimolar concentration of mannitol (control) for 24 h. High-glucose, but not high-mannitol, culture conditions resulted in significant increases in both Chrebpα and Chrebpβ mRNAs, indicating that high glucose stimulates ChREBP expression ex vivo. (C) To confirm the ex vivo findings, diabetic retinas were isolated for qRT-PCR analysis to evaluate ChREBP and MondoA gene expression. The qRT-PCR results show that retinas from 12-week-old db/db and Akita mice exhibit increased expression of both Chrebp and MondoA mRNAs. Statistical significance is indicated as * p < 0.05 and ** p < 0.01 (n = 4 per group).

Article Snippet: With lentiviral particles expressing ChREBP (Cat#: RC220626L2V OriGene Co., Rockville, MD, USA) was conducted using 100 μl of suspension.

Techniques: Expressing, RNAscope, Cell Culture, Concentration Assay, Control, Ex Vivo, Isolation, Quantitative RT-PCR, Gene Expression

Journal: Cells

Article Title: Glucose-Sensing Carbohydrate Response Element-Binding Protein in the Pathogenesis of Diabetic Retinopathy.

doi: 10.3390/cells14020107

Figure Lengend Snippet: Figure 3. Transgenic expression of constitutively active ChREBP in mouse rods leads to vision loss. (A) i-Cre-mediated recombination in rod photoreceptors of caChREBPRP transgenic mice resulted in deletion of eGFP and expression of FLAG-tagged caChREBP, detected by anti-FLAG antibody, shown in red. Nuclei are shown in blue. (B) Expression of caChREBP in rod photoreceptors leads to reduction in scotopic a- and b-wave amplitudes in caChREBPRP versus control (eGFPflox/wt-caChREBP or CghREBPf/wt) mice at postnatal day 35. Representative waveforms are shown on right. * p < 0.05, n = 3–6 per group.

Article Snippet: With lentiviral particles expressing ChREBP (Cat#: RC220626L2V OriGene Co., Rockville, MD, USA) was conducted using 100 μl of suspension.

Techniques: Transgenic Assay, Expressing, Control

Journal: Cells

Article Title: Glucose-Sensing Carbohydrate Response Element-Binding Protein in the Pathogenesis of Diabetic Retinopathy.

doi: 10.3390/cells14020107

Figure Lengend Snippet: Figure 4. Increased ChREBP activity in rods alters retinal proteomics and KEGG signaling in caChREBPRP mice. (A) Heatmap showing major altered proteins in caChREBPRP compared with eGFPflox/wt-caChREBP control (Control) retinas (n = 3–4 per group), highlighting significant changes in retinal protein expression. (B) Expression of constitutively active ChREBP (caChREBP) in rods leads to increase in TXNIP levels at postnatal day 35 (P35). Statistical significance is indicated by * p < 0.05, with n = 3–4 per group. (C) Major downregulated KEGG signaling pathways in caChREBP- expressing retinas are identified, showing significant pathway alterations due to sustained ChREBP activity in rods.

Article Snippet: With lentiviral particles expressing ChREBP (Cat#: RC220626L2V OriGene Co., Rockville, MD, USA) was conducted using 100 μl of suspension.

Techniques: Activity Assay, Control, Expressing, Protein-Protein interactions

Journal: Cells

Article Title: Glucose-Sensing Carbohydrate Response Element-Binding Protein in the Pathogenesis of Diabetic Retinopathy.

doi: 10.3390/cells14020107

Figure Lengend Snippet: Figure 5. Overexpression of ChREBP in human ARPE-19 cells. (A) The images of ARPE-19 cells overexpressing human ChREBP and control cells treated with the empty virus. The direct fluorescence emitted by GFP indicates successful infection and ChREBP expression. (B) The heatmap displays the major altered proteins in ARPE-19 cells with sustained ChREBP expression (n = 3–4 per group), highlighting significant protein expression changes due to ChREBP overexpression. (C) The results from the proteomic analysis were analyzed using the Shiny GO program to generate diagrams of altered KEGG pathways. Both decreased and increased pathways are shown, reflecting the impact of sustained ChREBP expression on cellular signaling networks in ARPE-19 cells.

Article Snippet: With lentiviral particles expressing ChREBP (Cat#: RC220626L2V OriGene Co., Rockville, MD, USA) was conducted using 100 μl of suspension.

Techniques: Over Expression, Control, Virus, Fluorescence, Infection, Expressing

Journal: Cells

Article Title: Glucose-Sensing Carbohydrate Response Element-Binding Protein in the Pathogenesis of Diabetic Retinopathy.

doi: 10.3390/cells14020107

Figure Lengend Snippet: Figure 6. ChREBP overexpression alters molecular function and affects cellular components of human ARPE-19 cells. (A) The molecular functions that are decreased or increased due to ChREBP overexpression in ARPE-19 cells are shown, highlighting the functional alterations induced by sustained ChREBP expression. (B) The cellular components affected by ChREBP expression in ARPE-19 cells are presented, demonstrating how ChREBP overexpression leads to changes in the cellular architecture and composition (n = 3–4 per group).

Article Snippet: With lentiviral particles expressing ChREBP (Cat#: RC220626L2V OriGene Co., Rockville, MD, USA) was conducted using 100 μl of suspension.

Techniques: Over Expression, Functional Assay, Expressing

Journal: European Journal of Medical Research

Article Title: Acute hyperglycemia exacerbates neuroinflammation and cognitive impairment in sepsis-associated encephalopathy by mediating the ChREBP/HIF-1α pathway

doi: 10.1186/s40001-024-02129-3

Figure Lengend Snippet: AH enhances neuroinflammation and up-regulates ChREBP expression in septic rats. a , b Hippocampal sections underwent IF staining at 48 h following CLP, followed by measurement of the number of cells positive for IBA1 and CD68. c – e Hippocampal pro-inflammatory cytokines levels (IL-1β, TNF- α , and HMGB1) were measured 48 h after CLP. n = 4. f – k Western blot assay indicates that AH upregulated the ChREBP, HIF-1α, HK2, PKM2, and iNOS expression 48 h after CLP. n = 3 in each group. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The lentiviral vectors for the knockdown of ChREBP (sh-ChREBP) or the non-silencing control shRNA (NC) were synthesized by Santa Cruz Biotechnology (cat. no. sc-3861, USA).

Techniques: Expressing, Staining, Western Blot

Journal: European Journal of Medical Research

Article Title: Acute hyperglycemia exacerbates neuroinflammation and cognitive impairment in sepsis-associated encephalopathy by mediating the ChREBP/HIF-1α pathway

doi: 10.1186/s40001-024-02129-3

Figure Lengend Snippet: High glucose enhances LPS-induced pro-inflammatory mediator production, mediated by the ChREBP/HIF-1α pathway in BV2 cells. BV2 cells underwent treatment with LPS (1 μg/mL) and 5.6 mM glucose (LG), 16.7 mM glucose (MG) or 25.0 mM glucose (HG). a – d Pro-inflammatory cytokines levels (IL-1β, TNF- α and HMGB1) in the supernatant and cell viability were ascertained at 6, 12, 24, and 48 h following LPS treatment. n = 4. e – g Twelve h after treatment, extracellular lactate content, as well as intracellular glycolytic and mitochondrial ATP levels, were assessed. n = 4. h – l Western blot assay indicates that high glucose upregulated the ChREBP, HIF-1α, HK2 and PKM2 expression 12 h after LPS treatment. m ChREBP, HIF-1α, HK2, and PKM2 relative mRNA levels ( n = 3 independent measurements). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The lentiviral vectors for the knockdown of ChREBP (sh-ChREBP) or the non-silencing control shRNA (NC) were synthesized by Santa Cruz Biotechnology (cat. no. sc-3861, USA).

Techniques: Western Blot, Expressing

Journal: European Journal of Medical Research

Article Title: Acute hyperglycemia exacerbates neuroinflammation and cognitive impairment in sepsis-associated encephalopathy by mediating the ChREBP/HIF-1α pathway

doi: 10.1186/s40001-024-02129-3

Figure Lengend Snippet: ChREBP Knockdown Prevents High Glucose-evoked Microglial Activation Following Exposure to LPS. BV2 cells were exposed to lenti-ChREBP–shRNA (sh-ChREBP) or lenti-NC (NC) for 48 h, and the resulting cell lines were then subjected to screening utilizing purinomycin (5 μg/mL), subsequent treatment with LPS (1 μg/mL) and/or 25.0 mM glucose (HG). a – c 12 h after treatment, extracellular lactate content and intracellular glycolytic and mitochondrial ATP levels were assessed. n = 4. d – k HIF-1α, HK2, PKM2, IL-1β, TNF- α and HMGB1 expression level. l ChREBP, HIF-1α, HK2, and PKM2 relative mRNA levels (n = 3 independent measurements). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The lentiviral vectors for the knockdown of ChREBP (sh-ChREBP) or the non-silencing control shRNA (NC) were synthesized by Santa Cruz Biotechnology (cat. no. sc-3861, USA).

Techniques: Knockdown, Activation Assay, shRNA, Expressing

Journal: European Journal of Medical Research

Article Title: Acute hyperglycemia exacerbates neuroinflammation and cognitive impairment in sepsis-associated encephalopathy by mediating the ChREBP/HIF-1α pathway

doi: 10.1186/s40001-024-02129-3

Figure Lengend Snippet: Elevated glucose levels amplify the ChREBP-mediated HIF-1α pathway, leading to microglial M1 polarization responding to LPS. Prior to treatment with 1 μg/mL LPS and/or 25.0 mM glucose for 12 h, BV2 cells were pre-exposed to KC7F2 (10 μM), which is an inhibitor of HIF-1α, for 24 h. a – c Twelve hours after treatment, extracellular lactate content and intracellular glycolytic and mitochondrial ATP levels were assessed. n = 4. d – k HIF-1α, HK2, PKM2, IL-1β, TNF-α, and HMGB1 expression levels were measured by Western blot. l ChREBP, HIF-1α, HK2, and PKM2 relative mRNA levels ( n = 3 independent measurements). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The lentiviral vectors for the knockdown of ChREBP (sh-ChREBP) or the non-silencing control shRNA (NC) were synthesized by Santa Cruz Biotechnology (cat. no. sc-3861, USA).

Techniques: Expressing, Western Blot