Journal: Cell reports

Article Title: Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression

doi: 10.1016/j.celrep.2020.108574

Figure Lengend Snippet: (A–C) DNA sequence motifs bound by WT SALL4A (A), a mutant lacking the 2nd zinc finger cluster (AΔZFC2) (B), or a mutant lacking the 4th ZFC (AΔZFC4) (C), discovered in universal PBM assays. The color bars above the position weighted matrices indicate the linear structure of SALL4, and the ZFCs are denoted by black ovals. (D) EMSA showing SALL4A shifts the AT-rich motif-containing oligos (lanes 1–3) but not when the motif is scrambled (lanes 4–6, gel cut for clarity); UC, unlabeled competitor probes. (E) SALL4-DNA complex is super-shifted by a SALL4 monoclonal antibody (lane 4) but not by mouse IgG isotype control (lane 5); mAb, SALL4 mouse antibody (Santa Cruz EE-30). (F) EMSA showing that SALL4-DNA complex was reduced or super-shifted in the presence of FLAG or SALL4 antibodies; rAb-1, SALL4 rabbit antibody (Cell Signaling D16H12); rAb-2, SALL4 rabbit antibody (Abcam ab57577). Lanes 8–10 show that the AΔZFC3 mutant binds to the same WT sequence, whereas binding by AΔZFC2 and AΔZFC4 mutants is abrogated. All EMSA reactions contain poly dI:dC competitor to reduce background binding. (G) Isothermal titration calorimetry (ITC) experiments showing purified SALL4 ZFC4 (amino acids 864–929) binds DNA oligos containing the WT WTATB motif (top) and not when the motif was mutated (bottom). All EMSA and ITC oligo sequences can be found in the .

Article Snippet: Here, we took advantage of the availability of a highly specific antibody against human SALL4 (Cell Signaling Technology, clone D16H12, lot 2) and performed the CUT&RUN assay , which is an in situ profiling of protein-DNA binding that eliminates the cross-linking step and generates reads with low background and more precise localization.

Techniques: Sequencing, Mutagenesis, Control, Binding Assay, Isothermal Titration Calorimetry, Purification

Journal: Cell reports

Article Title: Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression

doi: 10.1016/j.celrep.2020.108574

Figure Lengend Snippet: (A) Representative genomic tracks of three SALL4 CUT&RUN replicates (rep) and their isotype rabbit IgG control experiments; scale is 0–25. (B) The genomic distribution of SALL4 CUT&RUN peaks. (C) The top five HOMER motifs from de novo analysis of three CUT&RUN reps with their respective p values, resulting in the two composite motifs shown on right. (D) Bar graph showing the percentage of peaks containing Motif 2 in de novo (black bars) and direct (gray bars) analyses from SALL4 CUT&RUN reps.

Article Snippet: Here, we took advantage of the availability of a highly specific antibody against human SALL4 (Cell Signaling Technology, clone D16H12, lot 2) and performed the CUT&RUN assay , which is an in situ profiling of protein-DNA binding that eliminates the cross-linking step and generates reads with low background and more precise localization.

Techniques: Control

Journal: Cell reports

Article Title: Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression

doi: 10.1016/j.celrep.2020.108574

Figure Lengend Snippet: (A) Volcano plot showing genes that are down- or upregulated significantly after SALL4 KD with log2 fold change (FC) represented on the x axis; red circles denote differentially expressed genes with false discovery rate (FDR) of <0.05. (B) Number of differentially expressed genes after SALL4 KD (2,695) as well as those with annotated SALL4 peaks nearby (430) ( and ). (C) Bar graph representing number of up- and downregulated genes after SALL4 KD and their Gene Ontology (GO) molecular pathway analysis focusing on GO 0140110. (D) Volcano plot from (A) with genes encoding KDM proteins labeled; green circles denote genes containing SALL4 CUT&RUN peaks; the size of the circles corresponds to log 2 FC in expression. (E) Quantitative real-time PCR analysis of two SALL4 direct targets 40 h after SALL4 KD, summarized from either 3 or 4 independent experiments (primer sequences found in ); SCR, scrambled shRNA control. (F) EMSA showing that SALL4 binds KDM3A promoter region containing the WT AT-rich motif but not the mutated sequence

Article Snippet: Here, we took advantage of the availability of a highly specific antibody against human SALL4 (Cell Signaling Technology, clone D16H12, lot 2) and performed the CUT&RUN assay , which is an in situ profiling of protein-DNA binding that eliminates the cross-linking step and generates reads with low background and more precise localization.

Techniques: Labeling, Expressing, Real-time Polymerase Chain Reaction, shRNA, Control, Sequencing

Journal: Cell reports

Article Title: Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression

doi: 10.1016/j.celrep.2020.108574

Figure Lengend Snippet: (A) Western blotting of SNU398 liver cancer cell lysates at 40 h after SALL4 KD with gel cut for clarity; molecular weight marker is indicated on the right (left panel); Kd, kilodalton; summary of western blot densitometry from two separate SALL4 KD experiments of H3K9me2/3 protein expression (black bars) and KDM3A expression (gray bars) (right panel). (B) Western blotting of SNU398 cell lysates collected after either control DMSO or pomalidomide (Pom; 10 μM) treatments at the indicated time points. (C) Immunofluorescence staining of HP1 of SNU398 liver cancer cells transduced with SCR control or two shRNAs against SALL4; white scale bar denotes 33 μm; DAPI, 4’,6-diamidino-2-phenylindole DNA stain.

Article Snippet: Here, we took advantage of the availability of a highly specific antibody against human SALL4 (Cell Signaling Technology, clone D16H12, lot 2) and performed the CUT&RUN assay , which is an in situ profiling of protein-DNA binding that eliminates the cross-linking step and generates reads with low background and more precise localization.

Techniques: Western Blot, Molecular Weight, Marker, Expressing, Control, Immunofluorescence, Staining, Transduction

Journal: Cell reports

Article Title: Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression

doi: 10.1016/j.celrep.2020.108574

Figure Lengend Snippet:

Article Snippet: Here, we took advantage of the availability of a highly specific antibody against human SALL4 (Cell Signaling Technology, clone D16H12, lot 2) and performed the CUT&RUN assay , which is an in situ profiling of protein-DNA binding that eliminates the cross-linking step and generates reads with low background and more precise localization.

Techniques: Virus, SYBR Green Assay, Plasmid Preparation, Multiplex Assay, Gene Expression, Mutagenesis, shRNA, Software

Journal: Radiation research

Article Title: NATO BIODOSIMETRY STUDY

doi: 10.1667/RR3236.1

Figure Lengend Snippet: Methods Used by Contributing Laboratories

Article Snippet: Details of RNA isolation, cDNA synthesis and PCR parameters used by each laboratory are shown in . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Institution Chemistry No. genes Gene names Calibration and blinded test samples processed No. previous exercises Laboratory specialized in biodosimetry Method established (month) Method established for biodosimetry purposes (month) NATO samples processed with Time required for reported dose estimates (days) 1 SybrGreen 4 cdkn1a, gadd45, ddb2, bax separately 0 yes 144 6 priority 1.3 2 TaqMan 1 FDXR separately 0 no 120 2 priority 9.0 3 TaqMan 2 GADD45A, CDKN1A separately 0 no 72 1 when appropriate 16.0 SybrGreen nd nd nd 4 TaqMan 1 FDXR separately 0 yes 60 36 priority 1.2 TaqMan 1 FDXR together 5 TaqMan 2 DDB2, GADD45A together 1 yes 120 3 priority 0.8 6 TaqMan 1 FDXR together 0 yes 24 12 priority 0.3 7 CLPA 5 nd separately 0 yes 36 18 priority 0.3 8 CLPA 3 TNFSF9, PCNA, BAX separately 0 yes 60 60 when appropriate 9.0 Open in a separate window General Characteristics of Technical Procedures and Experiences for the Contributing Institutions are Shown table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Labor atory RNA isolation cDNA Synthesis RT-QPCR Isolation kit DNA digestion during Isolation Template eluted in QC Agilent-RIN// concentration// A260/280//A260/230// check DNA contamination Kit/MasterMix PCR protocol QC Kit Assays Cycles Platform Threshold Normalization Quantification method QC standard curve//slope//r2//18SrRNA-CT 1 QIAamp RNA Blood Mini Kit no RNAse-free water yes//no, Nano-Drop//yes// yes// adapted primer designs (intron flanking) Reverse Transcriptase Core kit (Eurogentec) Oligo dT priming 5′ -3′ assay LC Fast Start DNA Master SYBR Green kit ® (Roche Applied Science) GADD45A BAX DDB2 CDKN1A adapted for each gene LightCycler (Roche Applied Science) automated Human alu SX repeated elements ΔΔ CT approach yes// yes// yes// no: 5′ -3′ assay 2 Ambion Total RNA Extraction kit Turbo DNAse Elution Buffer Qubit used for RNA concentration High Capacity RNA-to-cDNA kit, AB 1×/37°C/60 min, 1×/95°C/5 min endogenous control -CT TaqMan Universal Master Mix II, AB Hs00169255_m1, GADDA45A Hs00171112_m1, CCNG1 Hs00172068_m1,DDB2 Hs00179935_m1, FHL2 Hs00180269_m1, BAX Hs00231069_m1, ATF3 Hs00234753_m1, MDM2 Hs00244586_m1, FDXR Hs00355782_m1, CDKN1A Hs00366278_m1, TNFRSF10B Hs00427214_g1, PCNA Hs00902787_m1, SESN1 Hs03645225_m1, PHPT1 1×/50°C/2 min 1×/ 95 °C/10 min 40×/ 95 °C/1 min 60°C/1 min ViiA7 AB automated 18S rRNA Δ Δ CT approach yes// yes// yes// yes 3 QIAamp RNA Blood Mini Kit RNase-free DNase-Set (Qiagen) RNAse-free water yes/yes; Nano-Drop QuantiTect Reverse Transcription Kit (QIAGEN) with integrated DNA removal 1.

Techniques: Isolation, Concentration Assay, SYBR Green Assay, RNA Extraction, Software, Multiplex Assay, Electrophoresis