Journal: Nature metabolism

Article Title: Genetic susceptibility to diabetic kidney disease is linked to promoter variants of XOR

doi: 10.1038/s42255-023-00776-0

Figure Lengend Snippet: a, Promoter luciferase assay performed on 293T cells with luciferase reporter constructs containing the Xor variants sequence upstream of the transcription start site; D-13, in normal glucose (NG; 5 mM) with control vector, mannitol (25 mM) or high glucose (HG; 30 mM) with D-13 vector (n = 3 biologically independent experiments; data are presented as mean values ± s.e.m.). b, Top, ChIP was carried out in kidney cortex tissue from diabetic B6 and D2 mice (n = 3 mice/group) using normal rabbit IgG antibodies, anti-STAT6 antibodies or anti-C/EBPβ antibodies, as indicated. Precipitated DNA was analysed by amplifying the PCR product in the Xor promoter, which is approximately 300 bp long. IgG served as a negative control. Bottom, a schematic of the C/EBPβ binding site in the sequence of the Xor promoter. c,d, Immunohistochemistry staining of C/EBPβ in diabetic B6 (c) and diabetic D2 (d) kidneys, showing nuclear staining in glomeruli (filled arrows) and some peritubular capillaries (open arrows). ×40 magnification; scale bars, 100 μm. e, Heatmap showing the genes that are differentially regulated by C/EBPβ in non-diabetic (n = 5) and diabetic D2 mice (n = 3); the arrow indicates Xdh as a top gene under the regulation of C/EBPβ in diabetes (P < 9.41 × 10−17). f, Cebpb mRNA in mGECs treated with a scrambled siRNA (Scrbl) or siRNA targeting Cebpb for 24 h (n = 7 biologically independent experiments, data are presented as mean values ± s.e.m.). g, Xor mRNA (bar graph) and protein (blot) expression in mGECs under the control condition NG or the HG condition, treated with scrambled or Cebpb-targeting siRNA for 24 h. h, Percentage of mGECs expressing CM-H2-DCFDA, a marker for ROS. mGECs were kept in NG or HG and were treated with scrambled or Cebpb siRNA for 24 h (n = 5 and n = 4 independent experiments in g and h, respectively; data are presented as mean values ± s.e.m.). To determine statistical significance, unpaired two-tailed Student’s t-tests were performed in f, and a one-way ANOVA with Tukey’s multiple-comparisons test was performed in a, g and h.

Article Snippet: Gene silencing RNA interference experiments in cells were performed after transfection of siRNA targeting Cebpb (Oligo Duplex (Locus ID 12608), OriGene) or with a scramble sequence, using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Luciferase, Construct, Sequencing, Control, Plasmid Preparation, Negative Control, Binding Assay, Immunohistochemistry, Staining, Expressing, Marker, Two Tailed Test

Journal: Cell reports

Article Title: Mapping Distinct Bone Marrow Niche Populations and Their Differentiation Paths

doi: 10.1016/j.celrep.2019.06.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: shRNA - TF500345 (Cebpb) , Origene , 4 unique shRNA.

Techniques: Recombinant, shRNA, Construct, Plasmid Preparation, Sequencing, Software

Journal: Brain : a journal of neurology

Article Title: A delta-secretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies.

doi: 10.1093/brain/awab062

Figure Lengend Snippet: Figure 1 d-Secretase-cleaved APP fragment selectively binds to CEBPB. (A) CEBPB-GFP was co-transfected with GST-tagged full- length APP, tau, a-syn or their d-secretase-truncated fragments into HEK293 cells. In 48 h, GST pull-down was performed and analysed with anti- GFP antibody (first panel). The expression of pulled down GST-tagged fragments (second panel) and constructs in whole cell lysates (third and fourth panels) were examined. The pulled down proteins were quantified relative to the expression level of each construct in cell lysates. Scatter plot, mean ± SEM (*P 5 0.05, one-way ANOVA). (B) c-Secretase and d-secretase-derived CTFs show high binding activity with GST-CEBPB.

Article Snippet: The shRNA plasmids were purchased from Origene: Cebpb Rat shRNA Plasmid (Locus ID 24253, Cat. TR710974); FE65 (APBB1) Human shRNA Plasmid Kit (Locus ID 322, Cat. TG306652).

Techniques: Transfection, Expressing, Construct, Derivative Assay, Binding Assay, Activity Assay

Journal: Brain : a journal of neurology

Article Title: A delta-secretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies.

doi: 10.1093/brain/awab062

Figure Lengend Snippet: Figure 2 APP C586-695 fragment interacts with CEBPB in the nucleus. (A) APP C586-695 strongly associates with CEBPB and Fe65, but Fe65 does not interact with CEBPB in 3xTg mice. Brain lysates from 12-month-old 3xTg, 3xTg/AEP KO and 3xTg/CEBPB + /– mice were immunoprecipitated with control IgG and anti-APP C586 (top) or control IgG and anti-Fe65 (bottom), analysed with anti-CEBPB, anti-Fe65 for APP C586 pulled-down groups (top) or anti-CEBPB, anti-APP C586 for Fe65 pulled-down groups (bottom). L.C. = light chain. (B) APP C586-695 activates CEBPB via enhancing its phosphorylation by activating phospho-ERK1/2 signalling. The nuclear and cytoplasmic fractions of 12-month-old

Article Snippet: The shRNA plasmids were purchased from Origene: Cebpb Rat shRNA Plasmid (Locus ID 24253, Cat. TR710974); FE65 (APBB1) Human shRNA Plasmid Kit (Locus ID 322, Cat. TG306652).

Techniques: Immunoprecipitation, Control, Phospho-proteomics

Journal: Brain : a journal of neurology

Article Title: A delta-secretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies.

doi: 10.1093/brain/awab062

Figure Lengend Snippet: Figure 3 APP C586-695 escalates CEBPB transcriptional activity. (A) OGD significantly enhances LGMN promoter luciferase activity in cells transfected with CEBPB, which was further promoted in the presence of APP C586-695. AEP luciferase promoter, CEBPB and APP CTF plasmids were co-transfected into HEK293 cells. Upon OGD treatment and recovery, cells were collected and subjected to the luciferase assay. Data represent mean ± SEM of three independent experiments; *P 5 0.05, **P 5 0.01, one-way ANOVA. (B) Real-time PCR analysis reveals that both APP C586-695 and AICD significantly enhance CEBPB transcriptional activity at the LGMN mRNA level, and APP C586-695 but not

Article Snippet: The shRNA plasmids were purchased from Origene: Cebpb Rat shRNA Plasmid (Locus ID 24253, Cat. TR710974); FE65 (APBB1) Human shRNA Plasmid Kit (Locus ID 322, Cat. TG306652).

Techniques: Activity Assay, Luciferase, Transfection, Real-time Polymerase Chain Reaction

Journal: Brain : a journal of neurology

Article Title: A delta-secretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies.

doi: 10.1093/brain/awab062

Figure Lengend Snippet: Figure 4 Inhibition of APP cleavage by d-secretase inhibits CEBPB activation by amyloid-b. (A) Primary cultured neurons were pre-treated with CP11 or dimethyl sulphoxide (DMSO) for 30 min then followed by the treatment of 2 mM pre-aggregated amyloid-b or DMSO for 24 h. Western blot (WB) analysis reveals that CP11 pretreatment diminished amyloid-b-induced d-secretase activation and the subsequent cleavage of APP and tau. Right: Quantification of the active band of AEP. Data represent mean ± SEM; *P 5 0.05, one-way ANOVA). (B) AEP in- hibitor CP11 inhibits the activity of d-secretase. Data represent mean ± SEM of three independent experiments; *P 5 0.05, t-test). (C)

Article Snippet: The shRNA plasmids were purchased from Origene: Cebpb Rat shRNA Plasmid (Locus ID 24253, Cat. TR710974); FE65 (APBB1) Human shRNA Plasmid Kit (Locus ID 322, Cat. TG306652).

Techniques: Inhibition, Activation Assay, Cell Culture, Western Blot, Activity Assay

Journal: Brain : a journal of neurology

Article Title: A delta-secretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies.

doi: 10.1093/brain/awab062

Figure Lengend Snippet: Figure 5 APP C586-695 but not APP FL stimulates CEBPB activities and promotes Alzheimer’s disease pathologies and cog- nitive disorders. (A) Hippocampal CA1 tissues from lentivirus-injected (GFP, APP C586-695-GFP, APP FL-GFP) wild-type (WT) or Cebpb + /–

Article Snippet: The shRNA plasmids were purchased from Origene: Cebpb Rat shRNA Plasmid (Locus ID 24253, Cat. TR710974); FE65 (APBB1) Human shRNA Plasmid Kit (Locus ID 322, Cat. TG306652).

Techniques: Injection

Journal: Brain : a journal of neurology

Article Title: A delta-secretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies.

doi: 10.1093/brain/awab062

Figure Lengend Snippet: Figure 6 Blockade of d-secretase APP cleavage abrogates APP SLA mutant-induced amyloid pathologies and cognitive dys- functions. (A) Hippocampal CA1 tissues from adeno-associated virus (AAV)-injected (Control, APPSLA, APPSLA N373A/N585A) wild-type or Cebpb + /– mice were analysed by western blot with various antibodies (n = 4 mice/group). Immunoblotting analysis shows that the protein levels of d-secretase and d-secretase-truncated APP, tau fragments are higher in APPSLA-expressing brain samples than control or APPSLA N373A/ N585A in wild-type mice and this difference is attenuated in Cebpb + /– mice. Right: Quantification of the active band of AEP. Data are box-and-

Article Snippet: The shRNA plasmids were purchased from Origene: Cebpb Rat shRNA Plasmid (Locus ID 24253, Cat. TR710974); FE65 (APBB1) Human shRNA Plasmid Kit (Locus ID 322, Cat. TG306652).

Techniques: Mutagenesis, Virus, Injection, Control, Western Blot, Expressing

Journal: Brain : a journal of neurology

Article Title: A delta-secretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies.

doi: 10.1093/brain/awab062

Figure Lengend Snippet: Figure 7 Clearance of APP C586-695 from 5xFAD diminishes CEBPB transcriptional activity and rescues cognitive functions. (A) APP C586 antibody administration reduces the levels of APP C586 (first panel). APP C586 antibody attenuates CEBPB-stimulated d-secre- tase activation and decreases the subsequent d-secretase-truncated APP and tau fragment generation compared with IgG injection group. Four- month-old 5xFAD mice were treated with control IgG or anti-APP C586 via intraperitoneal injection (twice/week, 2 mg/kg) for 3 months. Hippocampus lysates were analysed by western blot (n = 4 mice/group). Right: Quantification of the APP CTF, APP FL and amyloid-b. Box-and-

Article Snippet: The shRNA plasmids were purchased from Origene: Cebpb Rat shRNA Plasmid (Locus ID 24253, Cat. TR710974); FE65 (APBB1) Human shRNA Plasmid Kit (Locus ID 322, Cat. TG306652).

Techniques: Activity Assay, Activation Assay, Injection, Control, Western Blot