Journal:

Article Title: Transcriptional activation of the small GTPase gene rhoB by genotoxic stress is regulated via a CCAAT element

doi:

Figure Lengend Snippet: Members of the C/EBP family are not involved in the regulation of rhoB. (A) For gel retardation analysis the 32P-labeled oligonucleotide derived from the sequence harboring the CCAAT box essentially required for activation of the rhoB promoter by genotoxic stress was used (NF-YrhoB, 5′-GGCTTCCCATTGGGTGGCTAT-3′). Competition experiments were performed using 20-fold molar excess of either non-labeled NF-YrhoB oligonucleotide (specific competition) or an oligonucleotide containing the consensus sequence for binding of C/EBP (C/EBPa, 5′-TGCAGATTGCGCAATCTGCA-3′) (sc-2525; Santa Cruz). SComp, specific competition. (B) For band shift analysis the 32P-labeled C/EBP-specific oligonucleotide (C/EBPa) described in (A) was used. Competition experiments were done as described using either unlabeled C/EBPa (SComp) or NF-YrhoB-specific oligonucleotide. SComp, specific competition. (C) A second consensus sequence described to bind C/EBP proteins (26) was used for band shift analysis (C/EBPb, 5′-ATTCAATTGGGCAATCAG-3′). Specific competition (SComp) and competition with NF-YrhoB-specific oligonucleotide was done as described before.

Article Snippet: The following oligonucleotides were used for DNA binding analyses: NF-Y rhoB (5′-GGCTTCCC ATTGG GTGGCTAT-3′); AP-1-specific oligonucleotide (5′-AGTGG TGACTCA TCACT-3′); two different oligonucleotides for detection of binding of C/EBP proteins, C/EBP a consensus sequence (sc-2525; Santa Cruz) (5′-TGCAGATTGCG CAAT CTGCA-3′) and C/EBP b (5′-ATTCA ATTGG GCAATCAG-3′) ( 26 ); ATF/CREB (5′-AGAGATTGCC TGACGTCA GAGAGCTA-3′).

Techniques: Electrophoretic Mobility Shift Assay, Labeling, Derivative Assay, Sequencing, Activation Assay, Binding Assay

Journal: Cell Discovery

Article Title: CD36 + cancer-associated fibroblasts provide immunosuppressive microenvironment for hepatocellular carcinoma via secretion of macrophage migration inhibitory factor

doi: 10.1038/s41421-023-00529-z

Figure Lengend Snippet: a Isolation of CAF subtypes from HCC tumors via flow cytometry. b The average proportion of CAF subtypes in human or murine HCC tumors by flow cytometry. c Heatmap showed specific gene markers in CAF subtypes from murine and human HCC tumors. d , e The qPCR and ELISA assays showed MIF expression was higher in CD36 + CAFs among all fibroblasts. f The ELISA and western blot assays showed MIF expression was downregulated when CD36 was knockdown in CAFs. g The activity of reactive oxygen species pathway gene signatures in different CAF subclusters. h GSEA shows top enriched pathways in CD36 high vs CD36 low group and CD36 kd vs WT group. i – k Uptake of OxLDL and lipid peroxidation in CD36 + or CD36 kd CAFs was measured using fluorescently conjugated OxLDL and flow cytometry. l Human CD36 + CAFs treated with vehicle Ctrl, LDL (60 μg/mL), or OxLDL (30 or 60 μg/mL) for 24 h and then washed in PBS and incubated with BODIPY 581/591 C11 for the lipid peroxidation assay. m CD36 + or CD36 kd CAFs were treated with vehicle Ctrl, OxLDL (60 μg/mL), Toco (200 mM), SSO (100 mM), a combination of OxLDL (60 μg/mL) and Toco (200 mM), or a combination of OxLDL (60 mg/mL) and SSO (100 mM) for another 24 h. p38 phosphorylation (p-p38) was measured by flow cytometry, and the MFI of p-p38 was normalized to Ctrl. n The expression of p-p38 among CD36 + CAFs, CD36 kd CAFs and CD36 – CAFs from in vivo HCC murine models. o CD36 + CAFs were treated with vehicle Ctrl, OxLDL (60 μg/mL), SSO (100 mM), p38 inhibitor SB203580, a combination of OxLDL and SSO, or a combination of OxLDL and SB203580 for another 24 h. MIF secretion was measured by ELISA experiments, and the expression of MIF was nomalized to Ctrl. p CEBPA and CEBPD in CD36 + CAFs modulated the transcriptional expression of MIF by ChIP assays. Data are mean ± s.d. of n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student’s t test. n = 3 biological replica t es. Data shown as mean ± S.E.M., one-way ANOVA following multiple comparison test was used, *** P < 0.001, ** P < 0.01, * P < 0.05, and ns not significant.

Article Snippet: Cell lysates and supernatants were resolved by electrophoresis, transferred to a polyvinylidene fluoride membrane, and probed with antibodies against β-tubulin (Cat# 2128, Cell Signaling Technology), iNOS (Cat# ab178945, Abcam), CD36 (Cat# ab252922, Abcam), CEBPA (Cat# sc-365318, Santa cruz), CEBPD (Cat# sc-365546, Santa Cruz Biotechnology), MIF (Cat# ab187064, Abcam), p-p38 (Cat# ab195049, Abcam), p65 (Cat# ab32536, Abcam), SOX2 (Cat# ab92494, Abcam), OCT4 (Cat# ab181557, Abcam), STAT3 (Cat# ab68153, Abcam), or HLA-DRA (Cat# ab92511, Abcam).

Techniques: Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Knockdown, Activity Assay, Incubation, Peroxidation Assay, Phospho-proteomics, In Vivo, Comparison

Journal: Cell metabolism

Article Title: Aerobic Glycolysis Controls Myeloid-Derived Suppressor Cells and Tumor Immunity via a Specific CEBPB Isoform in Triple-Negative Breast Cancer

doi: 10.1016/j.cmet.2018.04.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LAP Over Expression Lentivirus LAP fragment with CMV promoter was cloned from LAP expression plasmid (plasmid #12557, Addgene) using KOD Xtreme hot start DNA polymerase (EMD Millipore).

Techniques: Control, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Lactate Assay, Cell Isolation, Gene Expression, shRNA, Plasmid Preparation, Software

Journal: Drug Design, Development and Therapy

Article Title: Tanshinone IIA Ameliorates Streptozotocin-Induced Diabetic Nephropathy, Partly by Attenuating PERK Pathway-Induced Fibrosis

doi: 10.2147/DDDT.S257734

Figure Lengend Snippet: Tan IIA down-regulates TGF-β1, TSP-1, Grp78 and CHOP expression in the renal tissues of the diabetic rats. ( A ) Immunohistochemical analysis of TGF-β1, TSP-1, Grp78 and CHOP expression in the renal tissues among five groups and the pooled data from ten sections for each group is summarized. Scale bar: 50 μm (×400). ( B, C ) The expression levels of Grp78 ( B ) and CHOP ( C ) from different treatment groups were determined by qRT-PCR. N = 10; * P <0.05 and ** P <0.01.

Article Snippet: The sections were incubated with primary antibodies against Grp78 (1:100), CHOP (1:50), TGF-β1 (1:100) and TSP-1 (1:100) overnight at 4°C, followed by incubating with biotinylated goat anti-rabbit IgG (1:200, # BA1003; Boster Biological Technology, Wuhan, China) in phosphate-buffered saline (PBS) for 2 h at room temperature.

Techniques: Expressing, Immunohistochemical staining, Quantitative RT-PCR