Journal: Blood

Article Title: Macrophage development from HSCs requires PU.1-coordinated microRNA expression

doi: 10.1182/blood-2011-02-335141

Figure Lengend Snippet: PU.1 regulates miR-146a in vivo. (A) MiR-146a expression in WT hematopoietic precursors and mature myeloid cells. miR-146a qRT-PCR was performed from FACS-purified HSCs (LSKs), common myeloid progenitors (CMPs; Lin−Sca-1−c-Kit+CD34+FcγRII/IIIlow), splenic granulocytes (Gra; Gr1+CD11b+), and splenic macrophages (Mac; Gr1−CD11b+). Data were compared with U6 RNA ± SD miR-146a and PU.1 mRNA expression in the indicated FACS-purified hematopoietic populations from WT and PU.1 KD animals. (B) MiR-146a was analyzed compared with U6 RNA, and PU.1 expression was determined relative to β-actin. **P < .001 by Student t test. (C) A 375-bp fragment containing the PU.1-binding site at 10 kb upstream of the genomic miR-146 locus was inserted in the pXP2 luciferase plasmid pXP2-(-)10kb. Mutation of the PU.1 motif (ctgaggaagt to ctgaccaagt) in the pXP2-(-)10kb construct, as indicated by the crossed-out PU.1 symbol in the pictogram, led to a marked reduction in reporter activity in transiently transfected myeloid RAW264.7 cells. Relative expression was determined 48 hours after transfection compared with pXP2 empty basal fluorescence activity (n = 3). **P < .001 by Student t test. (D) RAW264.7 cells carrying a stable PU.1 KD shRNA showed significantly lower miR146 promoter activity than plain RAW264.7 cells when transiently transfected with the WT pXP2-(-)10kb construct. Reporter activity of plain RAW264.7 cells was set to 100% (n = 3). **P < .001 by Student t test. (E) PU.1 expression in untreated and stably PU.1-shRNA–transfected RAW264.7 cells in Western blot normalized to levels of tubulin. Error bars indicate SD.

Article Snippet: A 375-bp genomic fragment located 10 kb upstream of miR-146a was PCR amplified (primer forward: 5′-ctcgaggagccggaatagaaggttcc-3′; primer reverse: 5′-aagcttaatgttaaattgaggtttttggtcg-3′) and subcloned into the pXP2 backbone (American Type Culture Collection no. 37577) via Xho I/ Hin dIII.

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Purification, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Construct, Activity Assay, Transfection, Fluorescence, shRNA, Stable Transfection, Western Blot

Journal: Communications Biology

Article Title: BRAF status modulates Interelukin-8 expression through a CHOP-dependent mechanism in colorectal cancer

doi: 10.1038/s42003-020-01263-y

Figure Lengend Snippet: a SNU1235, SNU1047, HT29, and LS180 cells lines were treated with the indicated concentration of drugs for 2 h and the presence of IL-8 was detected by RT-qPCR in all cell lines. Results were evaluated as ΔΔct of IL-8 relative to RPL19 and expressed as the ratio assuming the levels in the control as 1.0. Results of a representative experiment out of three independent experiments performed are shown. b Transcription factor binding regions in the IL-8 gene promoter retained in the different luciferase reporter plasmids. c Cell lines were co-transfected with 100 ng luc-reporter vector and 10 ng pRL-TK. Cells were harvested for luc assay 24 h post-transfection. pXP2 empty vector plasmid was used as a control. Results represent the average of three independent experiments. p -values indicate statistically significant differences ( p < 0.05 by 2-tailed Student’s t test) for the comparison between each reporter construct and the full promoter sequence (546-luc).

Article Snippet: The empty vector pXP2 plasmid in Escherichia coli (ATCC) was been extracted with QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions and used as a control.

Techniques: Concentration Assay, Quantitative RT-PCR, Control, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Comparison, Construct, Sequencing

Journal: Communications Biology

Article Title: BRAF status modulates Interelukin-8 expression through a CHOP-dependent mechanism in colorectal cancer

doi: 10.1038/s42003-020-01263-y

Figure Lengend Snippet: LS180 cell line was treated with fixed doses of dabrafenib (D) and trametinib (T) for 8 h, as indicated. a Whole LS180 lysate was immunoprecipitated with either anti-CHOP antibodies or control IgG and analyzed by WB using specific antibodies, as indicated; WB of the whole cell lysate are reported as control. LS180 chromatin was immunoprecipitated with anti-CHOP ( b ), anti-RNA Polymerase II ( c , d ) or anti-Acetyl-Histone H4 ( e ) antibodies or with control IgG and analyzed by RT-qPCR with primers specific for CHOP-binding region and TATA box of the IL-8 gene promoter. Results of a representative experiment out of three independent experiments performed are shown. f Cell lines were co-transfected with 100 ng luc-reporter vector and 10 ng pRL-TK. Cells were harvested for luc assay 24 h post-transfection and treated with fixed doses of D and T for 24 h, as indicated. pXP2 empty vector plasmid was used as a control. Results of a representative experiment out of three independent experiments performed are shown.

Article Snippet: The empty vector pXP2 plasmid in Escherichia coli (ATCC) was been extracted with QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions and used as a control.

Techniques: Immunoprecipitation, Control, Quantitative RT-PCR, Binding Assay, Transfection, Plasmid Preparation