Journal: Genes & Nutrition

Article Title: Increased protein intake affects pro-opiomelanocortin (POMC) processing, immune function and IGF signaling in peripheral blood mononuclear cells of home-dwelling old subjects using a genome-wide gene expression approach

doi: 10.1186/s12263-019-0654-6

Figure Lengend Snippet: Genes significantly regulated after intake of protein (FDR, q < 0.25)

Article Snippet: RT-qPCR was performed on an BioRad CFX96 (Bio–Rad Laboratories) with inventoried TaqMan gene expression assays for Killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 1 ( KIR2DL1, Hs 04961778_gH), KIR2DL4 (Hs00427106_m1), Killer cell lectin like receptor C3 ( KLRC3 , Hs01652462_m1), and C-C motif chemokine ligand 4 like 2 ( CCL4L2 , Hs04400556_m1) (Thermo Fisher Scientific).

Techniques: Sequencing, Binding Assay, Migration, Transduction, Ubiquitin Proteomics, Histone Deacetylase Assay, Activity Assay

Journal: Oncotarget

Article Title: Cross-talk between p21-activated kinase 4 and ERα signaling triggers endometrial cancer cell proliferation

doi: 10.18632/oncotarget.19188

Figure Lengend Snippet: (A-B) E 2 induces Pak4 mRNA and protein levels. Serum-starved Ishikawa, RL95-2 and MCF-7 cells were treated with 10 nM E 2 , and cells were harvested at the indicated time. (A) The levels of Pak4 mRNA were determined by qRT-PCR, using β-actin as an internal control. Values represent mean ± s.d. (n = 3). *** P <0.001 compared with control. (B) The protein levels of Pak4 were assessed by Western blot. (C) Ishikawa and RL95-2 cells were serum-starved for 24 h, and then treated with 10 nM E 2 for indicated times. The levels of p-Pak4ser 474 were measured by Western blot, using β-actin as a loading control. All experiments were carried out in triplicates.

Article Snippet: To stably silence Pak4 in RL95-2 cells, cells were transfected with two different shRNA constructs against human Pak4 or the control vector psiHIV-U6 (GeneCopoeia, Germantown, MD), and then selected with puromycin (0.5μg/mL, SIGMA Chemical, St Louis, MO, USA).

Techniques: Quantitative RT-PCR, Control, Western Blot

Journal: Oncotarget

Article Title: Cross-talk between p21-activated kinase 4 and ERα signaling triggers endometrial cancer cell proliferation

doi: 10.18632/oncotarget.19188

Figure Lengend Snippet: (A) Ishikawa and RL95-2 cells were treated with 10 nM E 2 for up to 90 min, and Western blot was used to detect p-AKT Ser 473 and total AKT levels. (B-C) RL95-2 cells were treated with E 2 for 60 or 90 min in the presence or absence of 20 μM LY 294002. Upper panel (B) p-AKT Ser 473 and (C) p-Pak4ser 474 levels were determined by western blotting, using β-actin as a loading control. Lower panel: Densitometric analysis of (B) p-AKT and (C) p-Pak4 in the immunoblots. Values represent mean ± s.d. (n = 3). * P < 0.05, *** P <0.001 compared with control, according to t-test. All experiments were carried out in triplicates.

Article Snippet: To stably silence Pak4 in RL95-2 cells, cells were transfected with two different shRNA constructs against human Pak4 or the control vector psiHIV-U6 (GeneCopoeia, Germantown, MD), and then selected with puromycin (0.5μg/mL, SIGMA Chemical, St Louis, MO, USA).

Techniques: Western Blot, Control

Journal: Oncotarget

Article Title: Cross-talk between p21-activated kinase 4 and ERα signaling triggers endometrial cancer cell proliferation

doi: 10.18632/oncotarget.19188

Figure Lengend Snippet: Immunofluorescence staining of (A) Pak4 and (B) p-Pak4 in RL95-2 cells treated with or without E 2 . Original magnification×400, bar=25μm. (C) Immunoblot analyses of Pak4 and p-Pak4 in subcellular protein fractions extracted from RL95-2 cells (T, total celllysate; C, cytoplasmic fraction; N, nuclear fraction). Cells were serum-starved for 24 h, and then treated with 10 nM E 2 , for indicated times.

Article Snippet: To stably silence Pak4 in RL95-2 cells, cells were transfected with two different shRNA constructs against human Pak4 or the control vector psiHIV-U6 (GeneCopoeia, Germantown, MD), and then selected with puromycin (0.5μg/mL, SIGMA Chemical, St Louis, MO, USA).

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Oncotarget

Article Title: Cross-talk between p21-activated kinase 4 and ERα signaling triggers endometrial cancer cell proliferation

doi: 10.18632/oncotarget.19188

Figure Lengend Snippet: (A) Left: Protein and mRNA levels of Pak4 were measured in wt Pak4-overexpressing Ishikawa cells by Western blot and qRT-PCR analysis, respectively; Right: ERα mRNA levels detected by qRT-PCR. Values are the mean ± SD from at least three independent experiments. (B) Left: Western blot and qRT-PCR of Pak4 levels in two different shPak4-transfected RL95-2 cells. Right: ERα mRNA levels detected by qRT-PCR. Values are the mean ± SD from at least three independent experiments. (C) Ishikawa cells were stably transfected with wt Pak4, ca Pak4, kinase-dead Pak4, or the control vector. (D) RL95-2 cells were stably transfected with two different shPak4 or the control vector. The ERE-Luc reporter plasmids were transfected into Ishikawa and RL95-2 cells 24 h before E 2 treatment, and luciferase assay was performed 48 h after E 2 addition. The mRNA levels of ERα target genes were determined by qRT-PCR. Cells were treated with 10 nM E 2 or vehicle for 48 h before RNA extraction. Values represent mean ± s.d. (n = 3), from three independent experiments. * P < 0.05, ** P <0.01, *** P <0.001 compared with control, according to t-test. (E) Schematic representation of the estrogen response element and the primers used for ChIP–qPCR. ERE: estrogen response element. TSS: transcription start sites. (F-G) A summary of ChIP-qPCR results for ERα binding in RL95-2 cells with the primer pairs shown in (E) . (F) shPak4 and control vector transfected RL95-2 cellswere treated with 10 nM E 2 for 48 h before DNA extraction. (G) RL95-2 cells were treated with 10 nM E 2 in the presence or absence of 1μM PF 3758309.

Article Snippet: To stably silence Pak4 in RL95-2 cells, cells were transfected with two different shRNA constructs against human Pak4 or the control vector psiHIV-U6 (GeneCopoeia, Germantown, MD), and then selected with puromycin (0.5μg/mL, SIGMA Chemical, St Louis, MO, USA).

Techniques: Western Blot, Quantitative RT-PCR, Transfection, Stable Transfection, Control, Plasmid Preparation, Luciferase, RNA Extraction, ChIP-qPCR, Binding Assay, DNA Extraction

Journal: Oncotarget

Article Title: Cross-talk between p21-activated kinase 4 and ERα signaling triggers endometrial cancer cell proliferation

doi: 10.18632/oncotarget.19188

Figure Lengend Snippet: (A) Soft agar colony assays of Pak4 knockdown, Pak4 inhibitor PF 3758309 treated RL95-2 cells and control cells. Cells were cultured in the medium with or without E 2 for 2 weeks. (B) RL95-2 cells were transfected with wt Pak4, ca Pak4, kinase-dead Pak4, or the control vector. Cells were cultured in the medium with 10 nM E 2 and 100 nM ICI 182,780 for 2 weeks. Representative images (left) were captured with an inverted phase contrast microscope (magnification, ×200). Columns (right), represent the number of colonies from three independent experiments, each in triplicates; values represent mean ± s.d.; * P < 0.05, ** P <0.01, *** P <0.001. (C) RL95-2 cells were stably transfected with shPak4 or control vector with GFP. The fluorescence images showing the transfection efficiency, as well as the decreased size of colonies in Pak4 knockdown cells compared with control cells. Original magnification, ×400. (D) MTT assay of Pak4 knockdown, Pak4 inhibitor PF 3758309 treated RL95-2 cells and control cells. Cells were either treated with E 2 , vehicle or left untreated as indicated. (E) RL95-2 cells were transfected with wt Pak4, ca Pak4, kinase-dead Pak4, or the control vector. Cells were treated with 10 nM E 2 , 10 nM E 2 + 100 nM ICI 182,780, or vehicle as indicated. All experiments were carried out in triplicates. (F) Cell-cycle profiles of shPak4 RL95-2 cells were assessed by FACS using DNA content profiles (left). Cells were either treated with E 2 , vehicle, or left untreated for 96 h before measurement. The percentages of cells in each compartment were calculated (right). Values represent mean ± s.d. (n = 3). * P < 0.05, ** P <0.01 compared with control, according to t-test. All experiments were carried out in triplicates.

Article Snippet: To stably silence Pak4 in RL95-2 cells, cells were transfected with two different shRNA constructs against human Pak4 or the control vector psiHIV-U6 (GeneCopoeia, Germantown, MD), and then selected with puromycin (0.5μg/mL, SIGMA Chemical, St Louis, MO, USA).

Techniques: Knockdown, Control, Cell Culture, Transfection, Plasmid Preparation, Microscopy, Stable Transfection, Fluorescence, MTT Assay

Journal: Oncotarget

Article Title: Cross-talk between p21-activated kinase 4 and ERα signaling triggers endometrial cancer cell proliferation

doi: 10.18632/oncotarget.19188

Figure Lengend Snippet: (A) Growth rates of tumors in nude mice inoculated with shPak4 RL95-2 cells or control cells. Values represent mean ± s.d. (n = 5). ** P <0.01 compared with control, according to t-test. (B) Immunohistochemical staining of Pak4 in control and shPak4 tumors.

Article Snippet: To stably silence Pak4 in RL95-2 cells, cells were transfected with two different shRNA constructs against human Pak4 or the control vector psiHIV-U6 (GeneCopoeia, Germantown, MD), and then selected with puromycin (0.5μg/mL, SIGMA Chemical, St Louis, MO, USA).

Techniques: Control, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Cross-talk between p21-activated kinase 4 and ERα signaling triggers endometrial cancer cell proliferation

doi: 10.18632/oncotarget.19188

Figure Lengend Snippet: Estrogen increases Pak4 expression and activation via PI3K/AKT pathway, the increased and activated Pak4 in turn enhances ERα transcriptional activity and cyclin D1 expression, which facilitates EC cell proliferation.

Article Snippet: To stably silence Pak4 in RL95-2 cells, cells were transfected with two different shRNA constructs against human Pak4 or the control vector psiHIV-U6 (GeneCopoeia, Germantown, MD), and then selected with puromycin (0.5μg/mL, SIGMA Chemical, St Louis, MO, USA).

Techniques: Expressing, Activation Assay, Activity Assay

Journal: Cell Reports Medicine

Article Title: UCPVax, a CD4 helper peptide vaccine, induces polyfunctional Th1 cells, antibody response, and epitope spreading to improve antitumor immunity

doi: 10.1016/j.xcrm.2025.102196

Figure Lengend Snippet: UCPVax induces hTERT-reactive CD4 + T cells in a broad range of HLA class II contexts (A) Treatment plan of UCPVax. UCPVax includes UCP2 and UCP4 pan-HLA-DR binding helper peptides derived from telomerase (hTERT) as previously described (Adotévi et al. ). Each helper peptide was emulsified in the adjuvant Montanide ISA-51 VG and injected subcutaneously. Patients received 6 weekly injections (priming) following by booster vaccinations for 8 weeks (boost) for a maximum of 12 months. Blood samples were collected for immune readouts as indicated (created with BioRender.com ). (B) Heatmap showing distribution of vaccine-induced UCP2- and UCP4-specific CD4 + T cells over time ( n = 60). The frequency of immune responders is shown at the top of the heatmap, intensity of response is shown at the bottom, and low and high responders were defined according to the median of IFNγ spots for each peptide (median spots: UCP2 = 84 and UCP4 = 63). The ∗ denotes patients evaluated with a mix of UCP2+UCP4. (C) Distribution of HLA-DR-B1 allele frequencies in patient responders to UCP2 ( n = 37) and UCP4 ( n = 24). (D) Heatmap representing HLA-DR-B1 allele expression in responders to UCP2 and UCP4 and in non-responders. (E) PHBR scores for UCP2 and UCP4. Boxplot represents median ± 1 st and 3 rd quartiles, Mann-Whitney test. (F) PHBR score according to the intensity of anti-UCP2 or UCP4 CD4 + T cell responses. (G) HLA-DR restriction. Histograms representing example of UCP2- (left) and UCP4- (right) specific responses with or without indicated MHC class I and II blocking antibodies measured by ex vivo ELISpot. Results represent mean of triplicates + SD. See also and and .

Article Snippet: Human IFN gamma ELISpot Set , Diaclone , Cat#856051020P.

Techniques: Binding Assay, Derivative Assay, Adjuvant, Injection, Expressing, MANN-WHITNEY, Blocking Assay, Ex Vivo, Enzyme-linked Immunospot

Journal: Cell Reports Medicine

Article Title: UCPVax, a CD4 helper peptide vaccine, induces polyfunctional Th1 cells, antibody response, and epitope spreading to improve antitumor immunity

doi: 10.1016/j.xcrm.2025.102196

Figure Lengend Snippet: Targeted CD4 + T cells with UCPVax promotes specific antibody response and epitope spreading (A) Schematic design of vaccine-induced antibody (Ab) response by ELISA in plasma. (B) UCP-specific IgG1 Ab titer (ng/mL) before (pre) and after priming vaccinations (post-vacc) ( n = 50). (C) Boxplot showing anti-UCP IgG1 titer Ab (ng/mL) pre- and post-vaccinations ( n = 50). Data are presented as median ± 1 st and 3 rd quartiles. Mann-Whitney test. (D) Graphs showing evolution of anti-UCP IgG response during vaccination in 10 representative patients. (E) Pie charts representing the distribution of patients with positive Ab response (POS; n = 31) on the left and without Ab response (NEG; n = 19) on the right according to the UCP CD4 + Th1 response by ELISpot; positive in red and negative in blue. (F) Overall survival (OS) according to anti-UCP Ab response ( n = 50). p value, two-tailed log rank test. (G) Schematic design of epitope-spreading assessment pre- and post-vaccination by IFNγ ELISpot after 6 days. IVS of PBMCs with mixture of MHC class I and II peptides derived from indicated tumor-associated antigens (TAAs) ( n = 41). (H) Representative IFNγ spot wells from two patients positive for epitope spreading. (I) Pie chart representing number of patients with (POS) or without (NEG) epitope-spreading induction. (J) Left, expansion of T cells against indicated TAA in the IFNγ ELISPOT assay. Right, distribution of specific T cells against class II epitope from TAA and class I peptides from hTERT. (K) Frequency of patients with epitope spreading according to anti-UCP CD4 + T cell response after vaccination. (L) Heatmap showing diversity of epitope-spreading response according to intensity of anti-UCP CD4 + T cells ( n = 25). (M) OS according to epitope spread response. p values, two-tailed log rank test.

Article Snippet: Human IFN gamma ELISpot Set , Diaclone , Cat#856051020P.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, MANN-WHITNEY, Enzyme-linked Immunospot, Two Tailed Test, Derivative Assay

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: MLL-AF4 ChIP-Seq Target Genes Are Upregulated in Primary B-ALLs (A) Wild-type MLL is proteolytically cleaved (dashed line) into N-terminal (MLL-N) and C-terminal (MLL-C) proteins. The t(4;11) breakpoint is marked by a black arrowhead labeled “bp.” The translocation fuses part of MLL-N in-frame with AF4-C (red box), and also produces a reciprocal AF4-MLL fusing AF4-N (violet box) with the rest of MLL. Antibody positions on the wild-type and fusion proteins are shown. A RUNX1 interaction domain at the C-terminal SET domain ( Huang et al., 2011 ) is indicated by blue shading. (B and C) ChIP-seq in RS4;11 cells across the HOXA cluster (B) and CDKN1B (C). The number of reads for peak summits was normalized by the total number of reads per track (set to 1 Gb for each track). Four different primer sets used for real-time PCR ChIP analysis are shown (red boxes) for the following amplicons: A9 , A10 , CDKN1B -A, and -B. (D) ChIP-seq in RS4;11 cells using antibodies to MLL-N, AF4-C, and H3K79Me2 produced an overlap at 603 target genes. (E) Comparison between the 603 RS4;11 target gene set from (D) and similar ChIP-seq data from SEM cells ( Guenther et al., 2008 ) produced a set of 491 common MLL-AF4 targets (see ). (F–I) The average expression of the 491 MLL-AF4 fusion target genes common in RS4;11 and SEM cells have significantly higher (p < 1e-6, two-tailed Wilcoxon test) expression levels than the nontarget genes in MLLr B-ALL patients in three different B-ALL clinical trials. (F) St. Jude Children’s Research Hospital, n = 20 MLLr patients ( Ross et al., 2003 ). (G) COG P9906 clinical trial, n = 21 MLLr patients ( Harvey et al., 2010 ). (H) ECOG E2993 clinical trial, n = 25 MLLr patients ( Geng et al., 2012 ). (I) The same data as in (H), split into t(4;11) versus other MLLr patient samples. Boxplots (F–H) represent the median values and error bars represent extreme maximum and minimum whisker values for each plot. Bar plots (I) are the mean and error bars represent SEM. See also and Figure S1 .

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: ChIP-sequencing, Labeling, Translocation Assay, Real-time Polymerase Chain Reaction, Produced, Comparison, Expressing, Two Tailed Test, Clinical Proteomics, Whisker Assay

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: MLL-AF4 Targets Genes in SEM Cells and Expression in ALL Patient Samples, Related to Figure 1 (A) MLL-AF4 target genes in SEM cells. ChIPseq analysis in SEM cells with antibodies to MLL-N, AF4-C and H3K79Me2 using data from ( Guenther et al., 2008 ) produced an overlap of 2490 target genes (list in ). (B–D) Average expression of the 491 target gene set (and the non target gene set) in MLL rearranged (MLLr) B-ALL patients from three B-ALL clinical trials expressed as bar plots with the error bars representing s.e.m. (standard error of mean). (B) St. Jude Children’s Research Hospital, n = 132 with 20 MLLr patients ( Ross et al., 2003 ). (C) COG P9906 clinical trial, n = 207 with 21 MLLr patients ( Harvey et al., 2010 ). (D) ECOG E2993 clinical trial, n = 191 with 25 MLLr patients ( Geng et al., 2012 ). (E–G) Average expression 491 target gene set expressed as barplots with the error bars representing s.e.m in clinical trials from St. Jude Children’s Research Hospital, BCR-ABL patients: 15, E2A-PBX1 patients: 18, MLLr patients: 20, RUNX1-ETV6 patients: 20, Other B-ALL patients: 28 (E), COG P9906 clinical trial, E2A-PBX1 patients: 23, MLLr patients: 21, RUNX1-ETV6 patients: 3, Other B-ALL patients: 155, (F) ECOG E2993 clinical trial, BCR-ABL patients: 78, E2A-PBX1 patients: 6, MLLr patients: 25 (t(4;11): 17, other MLLr: 8, Other B-ALL patients: 82.

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: Expressing, Produced, Clinical Proteomics

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: RUNX1 Is a Direct Target of MLL-AF4 and Is Specifically Upregulated in t(4;11) B-ALLs (A and B) ChIP-seq data in SEM (A) and in RS4;11 (B) cells across the RUNX1 locus using the antibodies as indicated. Reads were normalized as in Figure 1 . Gray bars highlight the positions of the P1 and P2 promoters as well as the +23 enhancer. Primer sets used for real-time PCR ChIP analysis are shown (red boxes). (C–H) The average expression of either HOXA9 (C–E) or RUNX1 (F–H) in three B-ALL clinical trials separated into different ALL subtypes as indicated. (C and F) St. Jude ALL patients ( Ross et al., 2003 ). (D and G) COG P9906 clinical trial ( Harvey et al., 2010 ). (E and H) ECOG E2993 clinical trial ( Geng et al., 2012 ). An asterisk indicates significantly lower average expression for the leukemia subtype relative to MLLr (C, D, F, and G) or relative to t(4;11) (E and H). A two-tailed Wilcoxon test was used to calculate p values, and p values for the different comparisons are in . See also , , and Figure S2 .

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: ChIP-sequencing, Real-time Polymerase Chain Reaction, Expressing, Clinical Proteomics, Two Tailed Test

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: ChIP across RUNX1 and Additional Gene Expression in ALL Patients, Related to Figure 2 (A) MLL-N, H3K4Me3 and H3K79Me2 ChIP in SEM (upper bar plots) and RS4;11 (lower bar plots) cells at a control region (1), HOXA9 (2), HOXA10 (3), CDKN1B (4 and 5), and across RUNX1 (6-13). Primer sets are as explained in and . (B–G) HOXA10 and CDKN1B are upregulated in primary B-ALLs with MLL1 rearrangements (MLLr). The average expression of either HOXA10 (B–D) or CDKN1B (E–G) was examined in B-ALL subtypes (including several non-MLL fusion proteins) using data from patients participating in 3 large B-ALL clinical trials. (B) and (E) St. Jude Children’s Research Hospital, ( Ross et al., 2003 ) separated into the following subtypes: BCR-ABL (blue 1), n = 15; E2A-PBX1 (green 2), n = 18; MLL rearrangements (MLLr), n = 20; ETV6-RUNX1 (teal 3), n = 20; Others (black 4), n = 28. (C) and (F) COG P9906 clinical trial, ( Harvey et al., 2010 ) separated into the following subtypes: E2A-PBX1 (green 2), n = 23; MLLr, n = 21; ETV6/RUNX1 (teal 3), n = 3; Others (black 4), n = 155. (D) and (G) ECOG E2993 clinical trial, ( Geng et al., 2012 ) separated into the following subtypes: BCR-ABL (blue 1), n = 78; E2A-PBX1 (green 2), n = 6; t(4;11), n = 17; Other MLLr, n = 8; Others (black 4), n = 82; normal preB cells (black 5), n = 3. An asterisk indicates significantly lower average expression for the leukemia subtype relative to MLLr (B,C,E,F) or relative to MLL-AF4 (D,G). HOXA10 and CDKN1B expression is not significantly different in MLLr versus MLL-AF4 samples (F and I, p > 0.1). Other p values for the different comparisons are in .

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: Gene Expression, Control, Expressing, Clinical Proteomics

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: MLL-AF4 Directly Regulates RUNX1 and Other Target Genes by Stabilizing AF9 and ENL Binding (A) MLL , MLL-AF4 , HOXA9, and RUNX1 real-time PCR expression in scrambled control siRNA-treated cells (black bars), MLL-AF4 siRNA-treated SEM (gray bars), and RS4;11 (white bars) cells. Data are the mean ± SD (error bars) of three independent knockdown experiments. In each individual experiment, control values were set to 1. (B and C) Western blots as indicated in SEM cells (B) or RS4;11 cells (C) treated with the siRNAs as indicated. Proteins were detected using the antibodies indicated except MLL-AF4, which was detected with an AF4-C antibody. (D) A summary of AF4 protein interactions. (E) MLL-N, AF4-C, AFF4, ENL, AF9, and Cyclin T1 ChIP + real-time PCR with scrambled control versus MLL-AF4 siRNA-treated SEM cells from (A). Values and error bars represent the mean ± SD of at least two independent ChIP experiments. Primer sets are as in Figure 1 B, 1C, and A.

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Control, Knockdown, Western Blot

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: In t(4;11) Cells, RUNX1 Is Highly Expressed and Has High Levels of ENL and AF9 Bound to the Locus (A) Real-time PCR quantification (see gene expression analysis in ) of HOXA9 (top), HOXA10 (middle), and RUNX1 (bottom) gene expression in patient cell lines. The cell lines analyzed are: RS4;11 (t-4;11), SEM (t-4;11), MV4-11 (t-4;11), THP-1 (MLL-AF9), NOMO-1 (MLL-AF9), MONO-MAC1 (MLL-AF9), KOPN-8 (MLL-ENL), ML-2 (MLL-AF6 and an MLL deletion), SHI-1 (MLL-AF6), RCH-ACV (normal MLL), CCRF-CEM (normal MLL), JURKAT (normal MLL), and K562 (normal MLL). Error bars represent the ±SD of two independent experiments. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; N.D., not detected. (B) Western blot of RUNX1 in the cell lines as described in (A) with a short exposure (top panel) and a long exposure (middle panel). (C) Western blot of nuclear extracts in the cell lines indicated and probed with the antibodies as indicated. (D) MLL-N, AF4-C, ENL, AF9, and Cyclin T1 ChIP in RS4;11 (dark red bars), SEM (spotted red bars), MV4-11 (bright red bars), THP-1 (black bars), KOPN-8 (blue bars), and CCRF-CEM (gray bars) patient cell lines. The control primer set is from a random gene-poor region on human chromosome 8; otherwise, primer sets are as indicated in B, 1C, and A. Error bars represent the ±SD of two independent experiments. See also Figure S3 .

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Control

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: High-Level RUNX1 Expression Is Important for t(4;11) Cell Growth and Correlates with a Poor Clinical Prognosis in MLL -Rearranged Leukemias (A) Real-time PCR expression of RUNX1 in THP-1 (MLL-AF9), SEM (t-4;11), and MV4-11 (t-4;11) cells treated with either a nontargeting control siRNA or two different RUNX1 siRNAs (#1 and #2). Data for THP-1#1 and SEM#1 are the mean ± SD of six independent experiments. The rest of the data are the mean ± SD of three independent experiments. Samples for gene expression analysis were taken the day of colony assay plating. (B) Representative western blots from samples in (A) probed with either RUNX1 or GAPDH antibodies. (C) Representative photomicrographs of THP-1 (left column) and SEM (right column) clonogenic cultures after treatment with either a nontargeting control (top row) or with RUNX1 siRNA#1 (bottom row). (D) Colony counts 14 days after plating. Data are the mean ± SD of either six independent experiments (THP-1#1 and SEM#1) or three independent experiments (the rest). Three replicates were plated per experiment. Control samples were set at 100% for each individual experiment. (E and F) Kaplan-Meier estimates of overall survival (OS) and relapse-free survival (RFS) based on minimal residual disease (MRD) measured at day 29 of the end-induction among 191 COG P9906 ( Harvey et al., 2010 ) ALL patients, log rank test p values. (G) A total of 67 MRD+ patients had higher average RUNX1 expression levels than 124 MRD− patients (p = 0.00746). (H) Among 17 MLLr patients, 9 patients that were MRD+ had significantly higher levels of RUNX1 expression than 8 MRD- patients (p = 0.0464, two-tailed Wilcoxcon test). (I) Among 174 non-MLLr B-ALL patients, 58 patients who were MRD+ had no significant increase in RUNX1 expression (p = 0.101, two-tailed Wilcoxon test). See also Figure S4 .

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Gene Expression, Colony Assay, Western Blot, Two Tailed Test

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: RUNX1 Expression Is Important for t(4;11) But Not MLL-ENL Growth, Related to Figure 5 (A) Real Time PCR expression of RUNX1 in SEM (t-4;11) or KOPN-8 (MLL-ENL) cells treated with either a non-targeting control siRNA or a RUNX1 siRNA (#1). Error bars represent the ± SD of three separate PCR reactions. (B and C) Cell counts of the control (blue line) or RUNX1 siRNA (red line) treated cells from (A) in SEM (B) or KOPN-8 (C) cells over ∼5 days. Error bars represent the ± SD of two independent experiments.

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: RUNX1 Interacts with the AF4-MLL Complex and Activates Gene Targets (A) RUNX1 ChIP-seq in SEM cells compared with MLL-C:H3K4Me3 and MLL-N:AF4-C:H3K79Me2 ChIP-seq. (B–D) Sample ChIP-seq tracks from SEM cells across MEF2C (B), ADAM10 (C), and SPI1/PU.1 (D). (E) Gene expression analysis by real-time PCR in SEM cells treated with two different RUNX1 siRNAs (gray bars, siRNA#1; white bars, siRNA#2). For each experiment, the PCR signal was quantified relative to control-treated cells. Results represent the mean ± SD of three independent knockdown experiments. (F) Western blots as indicated in SEM cells treated with a nontargeting control, RUNX1 siRNA#1, or a wild-type MLL siRNA. (G) RUNX1 protein complex interactions. RUNX1 can interact with a wild-type AF4 complex (interaction 1), a wild-type MLL complex (interaction 2), and potentially with an AF4-MLL complex (interaction 3). (H and I) Immunoprecipitation (IP) experiments using RS4;11 (H) and SEM (I) nuclear extracts. Extracts were IP’d with αIgG (lane 2), αAF4-N (lane 3), αRUNX1 (lane 4) or αMLL-C (lane 5), blotted and probed with the antibodies indicated. Input lanes represent 1% of the amount of extract used for the IPs. (J) A schematic of the MEF2D , JUNB , JUND, and SPI-1 (aka PU.1 ) loci showing the approximate location of PCR primer sets (open arrow heads) used for ChIP analysis. Black box indicates consensus RUNX1 binding motifs in the upstream regulatory region (URE) of SPI-1 ( Huang et al., 2008 ; Huang et al., 2011 ) and the first intron of MEF2D ( Pencovich et al., 2011 ). Gray box indicates exon 1 of MEF2D , JUND , JUNB, and SPI-1 . (K–M) ChIP analysis in SEM cells treated with a nontargeting control or RUNX1 siRNA#1 at the targets as indicated using antibodies to AF4-N (K), MLL-C (L), and RBBP5 (M). Error bars represent the ±SD of three separate PCR reactions. See also and Figure S5 .

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: ChIP-sequencing, Gene Expression, Real-time Polymerase Chain Reaction, Control, Knockdown, Western Blot, Immunoprecipitation, Binding Assay

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: RUNX1 ChIPseq and AF4-MLL Complex Data, Related to Figure 6 (A) Sample ChIP-seq tracks from SEM cells across RUNX1 , HOXA9 , MEF2D and JUND . (B) ChIP-seq overlap between the RUNX1 SEM cell target gene set versus the set of RUNX1 target genes from ( Pencovich et al., 2011 ). (C) Western blots for the proteins indicated in SEM cells treated with a scrambled control or an MLL-AF4 siRNA. Proteins were detected using the antibodies indicated except MLL-AF4, which was detected with an AF4-C antibody. (D) Gene expression analysis of selected genes in THP-1 cells treated with RUNX1 siRNA (siRNA#1). For each experiment, the PCR signal was quantified relative to the appropriate control treated cells. Results represent the average of three independent knockdown experiments, and error bars represent the standard deviation between experiments. (E) A schematic of protein complex interactions centering on the RUNX1 protein. RUNX1 can interact with a wild-type AF4 complex (interaction 1) through CyclinT1 ( Elagib et al., 2008 ; Jiang et al., 2005 ), a wild-type MLL-C complex (interaction 2) and potentially with an AF4-MLL complex (interaction 3). (F) Immunoprecipitation (IP) experiments using RS4;11 (t(4;11)), SEM (t(4;11)) and CCRF-CEM (wild-type MLL1) nuclear extracts. Extracts were IP’d with either αAF4-N (lane 3, 6 and 9) or a control αIgG (lane 2, 5 and 8) antibody, blotted and probed with the antibodies indicated. Lane 1,4 and 7 (Inputs) represents 1% of the amount of extract used for the IPs. AF4-MLL is indicated by a white arrowhead (AF4-MLL is 328 KDa, the white arrowhead represents the ∼194KDa Taspase 1 cleaved product) while wild-type AF4 is indicated by a black arrowhead (wild-type AF4 has a predicted size of 131 KDa but an apparent MW of 175 KDa). MLL-C is the Taspase 1 cleaved product of both AF4-MLL and wild-type MLL which is 134KDa but runs with an apparent MW of 180KDa. (G) Real Time PCR of MEF2D , JUNB and SPI-1 expression in SEM, RS4;11 and CCRF-CEM cells. (H) A schematic of the MEF2D , JUNB and SPI-1 (aka PU.1 ) loci showing the approximate location of PCR primer sets (open arrow heads) used for ChIP analysis. Black box = consensus RUNX1 binding motifs in the upstream regulatory region (URE) of SPI-1 ( Huang et al., 2008 ; Huang et al., 2011 ). and the first intron of MEF2D ( Pencovich et al., 2011 ). Grey box = exon1 of MEF2D , JUNB and SPI-1 . (I and J) ChIP analysis in SEM (black bars), RS4;11 (gray bars), CCRF-CEM (white bars) at the targets as indicated using antibodies to AF4-N (I) or RUNX1 (J). (K) Real Time PCR expression of AF4-MLLder4a and der4b ( Kumar et al., 2011 ) in SEM cells treated with a scrambled control (purple bars), an AF4-MLL siRNA (siRNA#10, blue bars) or an AF4-MLL siRNA (siRNA-K, orange bars) from ( Kumar et al., 2011 ). Error bars represent the ± SD of three separate PCR reactions. In each individual experiment, control values were arbitrarily set to 100. AF4-MLL siRNA sequences are in supplemental methods. (L) Western blots of the AF4-MLL knockdowns in (K) using the antibodies indicated. The apparent MW of AF4-MLL and MLL-C is explained in (F) above. (M) Western blots at day 4 and day 8 of SEM cells treated with both AF4-MLL siRNA-K and siRNA#10 at day 0, day 2, day 4 and day 7. Antibodies are as indicated. AF4-MLL is indicated by a white arrowhead while wild-type AF4 is indicated by a black arrowhead as explained in (F) above.

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: ChIP-sequencing, Western Blot, Control, Gene Expression, Knockdown, Standard Deviation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Binding Assay

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: MLL-AF4 Activates the RUNX1 Gene and the RUNX1 Protein Interacts with the AF4-MLL Complex and Activates Gene Targets (A) RUNX1 can interact with either coactivators or corepressors to cause gene activation or repression. In t(4;11) cells, RUNX1 can also interact with the AF4-MLL complex. (B) In t(4;11) leukemias, MLL-AF4 is expressed from one translocated chromosome, and the MLL-AF4 protein binds to and activates the RUNX1 gene by stabilizing AF9 and ENL binding. AF4-MLL is expressed from the other translocated chromosome, and the RUNX1 protein interacts with the AF4-MLL complex and binds to target genes.

Article Snippet: The following RUNX1 , GAPDH and HOXA9 Taqman primer/probe sets were used for the gene expression data in , and B: RUNX1 20X Taqman primer/probe set from ABI, cat# Hs00231079_m1 RUNX1 GAPDH 20X Taqman primer/probe set from ABI, cat# Hs03929097_g1 GAPDH HOXA9 Forward primer: AAAACAATGCCGAGAATGAGAGCG, Reverse primer: TGGTGTTTTGTATAGGGGGACC, FAM-TAMRA probe: CCCCATCGATCCCAATAACCCAGC The following SYBR green primer sets were used for the gene expression data in A, A, , B and 6G and : MLL , For: ACAGAAAAAAGTGGCTCCCCG, Rev: GCAAACCACCCTGGGTGTTA; MLL-AF4 (SEM cells), For: ACAGAAAAAAGTGGCTCCCCG, Rev: TATTGCTGTCAAAGGAGGCGG; MLL-AF4 (RS4;11 cells), For: TCAGCACTCTCTCCAATGGCAATAG, Rev: GGGGTTTGTTCACTGTCACTGTCC; AF4-MLLder4a , For: CAAGATCAGGCCCCTAGTGA, Rev: CCCATCTCCCACACATTTTC; AF4-MLLder4b , For: CAAGATCAGGCCCCTAGTGA, Rev: AGGGCTCACAACAGACTTGG; HOXA10 , For: CGCAACCACCCCAGCCAG, Rev: TTGTCCGCCGAGTCGTAGAGG; MEF2D , For: CTGAGCGTGCTATGTGACTGCG, Rev: TGGAGTGGTTGAAGATGATGAGTGC; JUNB , For: GGTCCAGGGAGCAGGCGG, Rev: CCAGTGTGGTTTGCGGCG; SPI-1 , For: CGGCTGGATGTTACAGGCGTG, Rev: TCGTGCGTTTGGCGTTGG; GAPDH , For: AACAGCGACACCCATCCTC, Rev: CATACCAGGAAATGAGCTTGACAA; Gene expression was normalized to GAPDH (either Taqman or SYBR green) by the ΔCT method.

Techniques: Activation Assay, Binding Assay

Journal: Nature Communications

Article Title: CD146-HIF-1α hypoxic reprogramming drives vascular remodeling and pulmonary arterial hypertension

doi: 10.1038/s41467-019-11500-6

Figure Lengend Snippet: HIF-1α induces CD146 expression in PASMCs. a , b Relative mRNA levels of CD146 in hPASMCs exposed to normoxia or hypoxia for indicated times. c CD146 protein expression in hPASMCs exposed to 21% or 1% O 2 for indicated times. d , e 293 T cells were transfected with siRNA targeting HIF1A , HIF2A , or TP53 for 24 h before exposed to 1% or 21% O 2 for another 24 h. CD146 expression at mRNA d or protein e level was measured. f , g Human PASMCs were treated with hypoxia, CoCl 2 (100 μM), DMOG (100 μM) or DFX (50 μM) for 24 h before CD146 mRNA f or protein g level was measured. h – k Human PASMCs were transfected with HIF-1α-expressing plasmid h , i or HIF-1α-siRNA j , k for 24 h before CD146 expression was measured. l Design of luciferase reporter vector containing human CD146 promoter that includes an HRE or a mutant HRE (mHRE). m Dual luciferase assay of putative HIF-1α-binding sites in CD146 promoter. The luciferase activity in pGL3 transfected construct was set as 1. n ChIP using ChIP-grade HIF-1α antibody and quantification of the enrichment of HIF-1α binding to CD146 promoter after hypoxia stimulation ( n = 3 per group). o , p Luciferase assay of HRE-WT reporter in human PASMCs transfected with HIF-1α siRNAs o , or treated with HIF-1α inhibitor digoxin or acriflavine. p Reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. n = 3 n , n = 4 a , b , d , f , h , j , or n = 5 m , o , p biological replicates for each group. All WB represent data from three c , e , g , i , k independent experiments. In all statistical plots, the results are expressed as mean ± s.e.m. * P < 0.05, * P < 0.01, *** P < 0.001. Two-tailed Student’s t test. Source data are provided as a Source Data file

Article Snippet: Flag-HIF-1α was purchased from Sino Biological Inc. Luciferase construct containing the human wild-type CD146 promoter 1 kb sequence upstream of translational start site was cloned into a pGL3 luciferase construct using Kpn I and Bgl III as insertion sites.

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Mutagenesis, Binding Assay, Activity Assay, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: CD146-HIF-1α hypoxic reprogramming drives vascular remodeling and pulmonary arterial hypertension

doi: 10.1038/s41467-019-11500-6

Figure Lengend Snippet: CD146 promotes HIF-1α transcriptional program through NF-κB. a , b HIF-1α mRNA a or protein b expression in murine PASMCs ( Cd146 +/+ and Cd146 −/− ) under normoxia or hypoxia. c Quantification of HIF-1α transcript in actinomycin D-treated PASMCs. d – f Luciferase assay of HRE-WT/HRE-Mut reporter d or Ca9 reporter e , or mRNA levels of Ca9 and Pai1 f in PASMCs ( Cd146 +/+ and Cd146 −/− ) under normoxia or hypoxia. g , h Luciferase assay of HRE-WT in Cd146 +/+ PASMCs transfected with HIF-1α-siRNA or in Cdd146 −/− PASMCs transfected with HIF-1α-expressing plasmid g , or treated with goxin h . i Dual luciferase assay of HIF1A promoter with the putative NF-κB-binding site or mutant site. j Quantification of ChIP assay of NF-κB binding to HIF1A promoter in human PASAMCs cultured under normoxic or hypoxic conditions in the presence or absence of NF-κB inhibitor BAY 11–7082. k – m HIF-1α mRNA k or protein l expression or reporter activity of HRE m in Cd146 −/− PASMCs transfected with CD146-expressing plasmid or p65-siRNA. n – p HIF-1α mRNA n or protein o expression or reporter activity of HRE p in Cd146 +/+ PASMCs treated with or without NF-κB inhibitor BAY 11–7082. q – s HIF-1α mRNA q or protein r expression or reporter activity of HRE s in human PASMCs transfected with CD146 dimer mutant-expressing plasmids. t – v HIF-1α mRNA n or protein o expression or reporter activity of HRE p in human PASMCs treated with anti-CD146 AA98 or mIgG (50 μg/ml). n = 3 j , n = 4 a , c , f , k , n , q , t , or n = 5 d , e , g , h , i , m , p , s , v biological replicates for each group. All WB represent data from three b , i , o , r , u independent experiments. In all statistical plots, the results are expressed as mean ± s.e.m. ** P < 0.01, *** P < 0.001. Two-tailed Student’s t test. Source data are provided as a Source Data file

Article Snippet: Flag-HIF-1α was purchased from Sino Biological Inc. Luciferase construct containing the human wild-type CD146 promoter 1 kb sequence upstream of translational start site was cloned into a pGL3 luciferase construct using Kpn I and Bgl III as insertion sites.

Techniques: Expressing, Luciferase, Transfection, Plasmid Preparation, Binding Assay, Mutagenesis, Cell Culture, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CD146-HIF-1α hypoxic reprogramming drives vascular remodeling and pulmonary arterial hypertension

doi: 10.1038/s41467-019-11500-6

Figure Lengend Snippet: CD146-HIF-1α axis promotes a synthetic phenotype in PASMCs. a – d Human a , b or mouse c , d PASMCs were transfected as indicated, and cultured under normoxic or hypoxic conditions, and cell proliferative ability and expression of indicated proteins were determined by CCK-8 assay a , c or WB b , d . e , f Human PASMCs were cultured under normoxic or hypoxic conditions in the presence of anti-CD146 AA98 or control mIgG (50 μg/ml). The cell proliferative ability and the expression of indicated proteins were determined by CCK-8 assay e or WB f . g , h Human g or mouse h PASMCs were transfected as in a or c . Cell migration was measured in a Transwell Boyden chamber. i Human PASMCs were treated as in e . Cell migration was measured in a Transwell Boyden chamber. j – m Human j , k or mouse l , m PASMCs were transfected as in a or c . The mRNA levels of contractile j , l and synthetic markers k , m were detected by real-time RT-PCR. n , o PASMCs were treated as in e . The mRNA levels of contractile n and synthetic markers o were detected by real-time RT-PCR. n = 4 j – o or n = 6 a , c , e , g – i biological replicates for each group. All WB represent data from three b , d , f independent experiments. In all statistical plots, the results are expressed as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant; by two-tailed Student’s t test. Source data are provided as a Source Data file

Article Snippet: Flag-HIF-1α was purchased from Sino Biological Inc. Luciferase construct containing the human wild-type CD146 promoter 1 kb sequence upstream of translational start site was cloned into a pGL3 luciferase construct using Kpn I and Bgl III as insertion sites.

Techniques: Transfection, Cell Culture, Expressing, CCK-8 Assay, Migration, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: CD146-HIF-1α hypoxic reprogramming drives vascular remodeling and pulmonary arterial hypertension

doi: 10.1038/s41467-019-11500-6

Figure Lengend Snippet: Disruption of CD146-HIF-1α in SMCs attenuates hypoxic PH. a Schematic of hypoxia-induced PH. b Left, WB analysis of CD146, HIF-1α and p-p65 in PASMCs from CD146 SMC-KO and CD146 WT mice. Right, relative expression of CD146, HIF-1α and p-p65 ( n = 3 biological replicates for each group). c The mRNA levels of Ca9 and Pai1 in PASMCs were measured by real-time RT-PCR (n = 5 biological replicates for each group). d – h RVSP d , TPVRI e , SAP f , RV/(LV + S) g and body weight h in mice after 4 weeks of hypoxia ( n = 10 mice per group). i Echocardiographic (PAAT, RVID, RVWT, and TAPSE) measurements and images. VTI, velocity time integral. j Echocardiography measurements were performed on CD146 SMC-KO and CD146 WT mice after exposure to hypoxic conditions for 4 weeks to determine PAAT, RVID, RVWT, TAPSE, CO, and CI ( n = 10 mice per group). k Representative images of H&E and immunofluorescent staining of PAs (20–50 μm or 51–100 μm in diameter) stained with αSMA (green). Scale bar, 50 μm. l Quantification of vascular medial thickness for images in k ( n = 5 mice per group, five PAs per mouse). m Quantification of PAs with > 50% luminal stenosis ( n = 5 mice per group, five PAs per mouse). n Proportion of non-, partially-, or fully- muscularized pulmonary arterioles (20–50 μm in diameter) from hypoxia-treated mice ( n = 8 mice per group). o Left, immunofluorescence staining of lung samples for αSMA (red) and PCNA (green). Right, quantification of the relative number of PCNA + /αSMA + cells. p Left, TUNEL staining (green) with DAPI nuclear staining (blue) in small PAs. Right, quantification of the number of TUNEL + cells. n = 4 o , p mice per group, three PAs per mouse. Scale bar, 50 μm. In all statistical plots, the results are expressed as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant; by one-way ANOVA with Bonferroni post hoc analysis d – h , j or two-tailed Student’s t test b , c , l – p . All WB represent data from three b independent experiments. Source data are provided as a Source Data file

Article Snippet: Flag-HIF-1α was purchased from Sino Biological Inc. Luciferase construct containing the human wild-type CD146 promoter 1 kb sequence upstream of translational start site was cloned into a pGL3 luciferase construct using Kpn I and Bgl III as insertion sites.

Techniques: Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, TUNEL Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CD146-HIF-1α hypoxic reprogramming drives vascular remodeling and pulmonary arterial hypertension

doi: 10.1038/s41467-019-11500-6

Figure Lengend Snippet: Targeting CD146-HIF-1α axis attenuates hypoxic PH in mice. a Schematic of CD146-targeted therapy in hypoxia-induced PH in mice. b Left, WB analysis of CD146 and HIF-1α in PASMCs isolated from anti-CD146- or mIgG-treated mice. Right, relative expression of CD146 and HIF-1α ( n = 3 biological replicates for each group). c mRNA levels of Ca9 and Pai1 in PASMCs were detected by real-time RT-PCR ( n = 5 biological replicates for each group). d – g RVSP d , TPVRI e , SAP f and RV/(LV + S) g in anti-CD146- or mIgG-treated mice after 4 weeks of hypoxia ( n = 10 mice per group). h Echocardiographic (PAAT, RVID, RVWT, and TAPSE) measurements and images. i Echocardiography measurements were performed on anti-CD146- or mIgG-treated mice to determine PAAT, RVID, RVWT, TAPSE, CO and CI ( n = 10 mice per group). j Representative images of H&E and immunofluorescent staining of PAs (20–50 μm or 51–100 μm in diameter) stained with αSMA (green). Scale bar, 50 μm. k Quantification of vascular medial thickness for images in j ( n = 5 mice per group, five PAs per mouse). l Quantification of PAs with >50% luminal stenosis ( n = 5 mice per group, five PAs per mouse). m Proportion of non-, partially-, or fully muscularized pulmonary arterioles (20–50 μm in diameter) from hypoxia-treated mice ( n = 8 mice per group). n Left, immunofluorescence staining of lung samples for αSMA (red) and PCNA (green). Right, quantification of the relative number of PCNA + /αSMA + cells. o Left, TUNEL staining (green) with DAPI nuclear staining (blue) in small PAs. Right, quantification of the number of TUNEL + cells. n = 4 n , o mice per group, three PAs per mouse. Scale bar, 50 μm. All WB represent data from three b independent experiments. In all statistical plots, the results are expressed as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant; by one-way ANOVA with Bonferroni post hoc analysis d – g , i or two-tailed Student’s t test b , c , k – o . Source data are provided as a Source Data file

Article Snippet: Flag-HIF-1α was purchased from Sino Biological Inc. Luciferase construct containing the human wild-type CD146 promoter 1 kb sequence upstream of translational start site was cloned into a pGL3 luciferase construct using Kpn I and Bgl III as insertion sites.

Techniques: Isolation, Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, TUNEL Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CD146-HIF-1α hypoxic reprogramming drives vascular remodeling and pulmonary arterial hypertension

doi: 10.1038/s41467-019-11500-6

Figure Lengend Snippet: Targeting CD146-HIF-1α axis attenuates MCT-induced PH in rats. a Schematic of CD146-targeted therapy in MCT-induced PH in rats. b – e RVSP b , TPVRI c , SAP d and RV/(LV + S) e in anti-CD146- or mIgG-treated rats after 5 weeks of MCT ( n = 10 rats per group). f Echocardiographic (PAAT, RVID, RVWT, and TAPSE) measurements and images. g Echocardiography measurements were carried out on anti-CD146- or mIgG-treated rats after 5 weeks of MCT to determine PAAT, RVID, RVWT, TAPSE, CO, and CI ( n = 10 rats per group). h Representative images of H&E and immunofluorescent staining of PAs (20–50 μm or 51–100 μm in diameter) stained with αSMA (green). Scale bar, 50 μm. i Quantification of vascular medial thickness for images in h ( n = 5 rats per group, five PAs per rat). j Quantification of PAs with >50% luminal stenosis ( n = 5 rats per group, five PAs per rat). k Proportion of non-, partially-, or fully- muscularized pulmonary arterioles (20–50 μm in diameter) from MCT-treated rats ( n = 8 rats per group). l Quantification of the relative number of PCNA + /αSMA + cells ( n = 4 rats per group, three PAs per rat). m Quantification of the number of TUNEL + cells ( n = 4 rats per group, three PAs per rat). In all statistical plots, the results are expressed as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant; one-way ANOVA with Bonferroni post hoc analysis b – e , g or two-tailed Student’s t test i – m . Source data are provided as a Source Data file

Article Snippet: Flag-HIF-1α was purchased from Sino Biological Inc. Luciferase construct containing the human wild-type CD146 promoter 1 kb sequence upstream of translational start site was cloned into a pGL3 luciferase construct using Kpn I and Bgl III as insertion sites.

Techniques: Staining, TUNEL Assay, Two Tailed Test

Journal: Cancer discovery

Article Title: Tumor microenvironment remodeling enables bypass of oncogenic KRAS dependency in pancreatic cancer

doi: 10.1158/2159-8290.CD-19-0597

Figure Lengend Snippet: A, Schematic graphs of genetic alleles in the iKPC genetically engineered mouse model, and control of KRAS* expression by Doxycycline (DOX). B, Relative total Kras gene expression level in iKPC-1 orthotopic allograft tumors with or without 24-hour DOX feeding (n=4 tumors for each group). C, Activation of KRAS* major downstream MEK/ERK pathway in iKPC-1 orthotopic allograft tumors with or without 24-hour DOX feeding (n=5 tumors for each group). D, Schematic diagram of screening strategy. E, Schematic experimental design of KRAS* bypass in vivo. F, Single ORF validation of top 10 candidates to bypass KRAS* dependency in vivo. G, HDAC5 promotes KRAS*-independent tumor growth in 5 different iKPC cell lines. Each iKPC cell line overexpressing GFP or HDAC5 was subcutaneously transplanted in nude mice at 500,000 cells per injection. Five mice with GFP-overexpressed (OE) iKPC cells were given Doxycycline water (ad lib) to activate KRAS* expression as a positive control group; five mice with GFP-OE iKPC cells and five mice with HDAC5-OE iKPC cells were given normal water to extinct KRAS* expression as negative control and experimental group, respectively. Tumor sizes were measured on the days indicated after transplantation. H and I, Tumor volume analysis of nude mice subcutaneously transplanted with GFP-, HDAC5- or HDAC5D-OE iKPC-3 cells (H) or iKPC-1 cells (I). Mice were given normal water to extinct KRAS* expression. J, BLI imaging of nude mice orthotopically transplanted with GFP-, HDAC5- or HDAC5D-OE iKPC-1 cells with luciferase reporter. K, The Kaplan–Meier survival analysis of nude mice orthotopically transplanted with GFP-, HDAC5- or HDAC5D-OE iKPC-5 cells. The Gehan-Breslow-Wilcoxon tests were performed to calculate the p values. L, Pancreas weight analysis from nude mice orthotopically transplanted with GFP-, HDAC5- or HDAC5D-OE iKPC-3 cells at day 53 after KRAS* extinction. M, Summary of all the in vivo KRAS* bypass experiments comparing the bypass efficiency driven by GFP, HDAC5 and HDAC5D in iKPC cells. N, H&E staining and IHC staining of pERK, pS6 and Ki67 in HDAC5 escapers and iKPC tumors derived from nude mice. The 40x images are not necessarily closeups of the 20x slides. O, The 3-D colony formation assay of GFP-, HDAC5- or HDAC5D-OE iKPC-1 and iKPC-5 cells after KRAS* extinction in Matrigel culture under normoxia or hypoxia conditions. KRAS*-expressing cells were used as positive control. P, Upregulated pathways in HDAC5 escaper cells (n=5) versus iKPC cells (n=4) by GSEA analysis of RNA-seq data. For B and L, data are represented as mean ± SEM. For B, G-I, L and M, two-tailed unpaired t tests were performed to calculate the p values.

Article Snippet: RNA extraction, qRT-PCR, mRNA sequencing and GSEA analysis RNA Extraction Kit (Qiagen) was used to extract RNA from tumor and cell samples.

Techniques: Control, Expressing, Gene Expression, Activation Assay, In Vivo, Biomarker Discovery, Injection, Positive Control, Negative Control, Transplantation Assay, Imaging, Luciferase, Staining, Immunohistochemistry, Derivative Assay, Colony Assay, RNA Sequencing, Two Tailed Test

Journal: Cancer discovery

Article Title: Tumor microenvironment remodeling enables bypass of oncogenic KRAS dependency in pancreatic cancer

doi: 10.1158/2159-8290.CD-19-0597

Figure Lengend Snippet: A, Exploration of HDAC5 targets by overlapping 3 profiling datasets: 5589 HDAC5 binding genes from ChIP-seq data, 131 differentially expressed genes (DEGs) in immune pathways after knockdown of HDAC5 comparing to scramble control in HDAC5-driven escaper cells, and 3758 downregulated genes in HDAC5-driven escaper cells comparing to iKPC cells. Seventeen candidate genes were filtered out and ranked by p-values in the 2 RNA-seq datasets from low to high. Top 5 candidates are represented. B, Comparison of Socs3 expression in iKPC cells and HDAC5-driven escaper cells. C and D, Upregulation of Socs3 expression after knockdown of HDAC5 in HDAC5-driven escapers at mRNA level (C) and protein level (D). E, Comparison of Socs3 expression in iKPC cells overexpressing HDAC5D and HDAC5. F, Binding sites of HDAC5 on Socs3 promoter and gene body region from ChIP-seq data. P1-P4 are primers designed for ChIP-q-PCR validation. G, ChIP-q-PCR validation of the binding of HDAC5 on Socs3 promoter and gene body regions. H, Gene expression of neutrophil- and macrophage-attracted chemokines after knockdown of Socs3 in iKPC cells. I, Validation of interactions between HDAC5 and NFIX or MEF2D by co-IP/WB analysis. J, HDAC5-ChIP-q-PCR analysis of HDAC5 escaper cells with scramble control and with knockdown of Nfix or Mef2d. Data are represented as mean ± SEM, and two-tailed unpaired t tests were performed to calculate the p values. K, Heatmaps of overall peak locations relative to the TSS for H3K4me3, H3K9ac and H3K27ac in GFP-OE and HDAC5-OE iKPC-1 samples as well as in HDAC5-FLAG escaper #1 cells with scramble control and HDAC5 knockdown (shH5-1). L, Schematic display of the overlapped genes that are bound by HDAC5 and marked by H3K27ac. M, GSEA analysis of the overlapped genes that are bound by HDAC5 and marked by H3K27ac. N, Histone acetylation marker status at Socs3 loci in the two comparison groups. For B, C, E, G and H, data are represented as mean ± SD, and two-tailed unpaired t tests were performed to calculate the p values.

Article Snippet: RNA extraction, qRT-PCR, mRNA sequencing and GSEA analysis RNA Extraction Kit (Qiagen) was used to extract RNA from tumor and cell samples.

Techniques: Binding Assay, ChIP-sequencing, Knockdown, Control, RNA Sequencing, Comparison, Expressing, Biomarker Discovery, Gene Expression, Co-Immunoprecipitation Assay, Two Tailed Test, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TNFR2 Signaling Enhances Suppressive Abilities of Human Circulating T Follicular Regulatory Cells.

doi: 10.4049/jimmunol.2100323

Figure Lengend Snippet: FIGURE 1. Gating strategy for each T cell subset and expression of TNFR2 on Tfr cells. (A) The gating strategy for Tfr, Treg, and Tconv cells in flow cytometry. Treg, Tfr, and Tconv cells were defined as CXCR5−CD25highCD127lowCD41CD14−, CXCR51CD25highCD127lowCD41CD14−, and CXCR5−CD25lowCD127highCD41CD14−, respectively. (B) Expression of TNFR2 relative to 18S rRNA in flow-sorted Tconv and Tfr cells determined by qPCR. Relative expression levels of TNFR2 were normalized to 18S rRNA and compared with the values obtained for Tconv cells. The sample of Tconv cells was assigned the arbitrary value of 1.0. Analysis for each sample was carried out in triplicate. Bars show mean ± SD of each group (n 5 3 per group). (CE) Cell surface expression of TNFR2 on Tconv, Treg, and Tfr cells was analyzed by flow cytometry. The histogram of TNFR2 staining is shown in (C). MFI of TNFR2 is indicated in (D). The positivity ratio is shown in (E). (F) Specificity of MR2-1 to TNFR2 on cell surface. The Jurkat cells overexpressing TNFR1 or TNFR2 were incubated on ice with MR2-1 or isotype in the presence of fluorescent dyeconjugated anti-mouse IgG1 (second Ab). The binding activity of MR2-1 was analyzed by flow cytometry, and the histogram is shown. (G) Agonistic function of H398. The synovial fibroblasts from a patient with RA were incubated with 1 mg/ml H398, isotype, or 5 ng/ml sTNF for 24 h. The concentration of IL-8 in the supernatant was measured by ELISA. Bars show mean ± SD of each group (n 5 3 per group). *p < 0.05 (two-tailed Student t test).

Article Snippet: The culture supernatants were collected, and the concentration of Ig was analyzed using the Human IgM ELISA Quantitation Set, Human IgA ELISA Quantitation Set, and Human IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX).

Techniques: Expressing, Flow Cytometry, Staining, Incubation, Binding Assay, Activity Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TNFR2 Signaling Enhances Suppressive Abilities of Human Circulating T Follicular Regulatory Cells.

doi: 10.4049/jimmunol.2100323

Figure Lengend Snippet: FIGURE 5. The suppressive function of Tfr cells against Tfh and B cells induced by TNFR2 stimulation. (A and B) Precultured Tfr cells were cocultured with CellTrace-labeled Tfh cells and PFA-fixed allo-PBMCs in the presence of IL-2 and OKT3 for 4 d. Cultured cells were harvested, the cell proliferation of Tfh cells was analyzed by flow cytometry (A), and the proportion of proliferated Tfh cells is indicated (B). (C) Tfr and Treg cells were precultured and used for suppression assay as in (B). The E:T ratio (Treg or Tfr cells/Tfh cells) was altered from 1:2 to 1:16. In this figure, a one-tailed Student t test was per- formed. (DG) Tfr cells were precultured in the same way as in (A)(C) [in (D), Tconv and Treg cells were also precultured as Tfr cells]. Then, freshly sorted Tfh and naive B cells were cocultured with pretreated Tfr cells [in (D), Tconv or Treg or freshly sorted Tfr cells were also cocultured] in the presence of staphylococcal enterotoxin B. After 7 d, the culture supernatant and cells were collected. The concentration of IgM in the culture supernatant was analyzed by ELISA (D). The proportion of plasma cells (E) and memory B cells (F) in total B cells was analyzed by flow cytometry. The cell proliferation of B cells (G) was analyzed by flow cytometry using CellTrace assay. The proportion of proliferated B cells per total B cells is indicated in (F). Bars show mean ± SD of each group (n 5 3 per group). *p < 0.05 [two-tailed Student t test except for (C)].

Article Snippet: The culture supernatants were collected, and the concentration of Ig was analyzed using the Human IgM ELISA Quantitation Set, Human IgA ELISA Quantitation Set, and Human IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX).

Techniques: Labeling, Cell Culture, Flow Cytometry, Suppression Assay, One-tailed Test, Concentration Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Two Tailed Test

Journal: Journal of Virology

Article Title: The Alpha/Beta Interferon Receptor Provides Protection against Influenza Virus Replication but Is Dispensable for Inflammatory Response Signaling ▿ †

doi: 10.1128/JVI.01595-09

Figure Lengend Snippet: TLR3, PKR, and Stat1 but not IFN-β mRNA levels are decreased in the absence of the IFN-α/β receptor during influenza virus infection. WT, IFN-γR−/−, IFN-αβR−/−, or IFN-αβγR−/− MEFs were mock infected or infected with the WSN, r1918, or VN1203 strain of influenza virus at an MOI of 2 PFU/cell. At 24 h p.i., total RNA was isolated from the cells and reverse transcribed to generate cDNA. Quantitative RT-PCR was used to determine the amount of TLR3 (A), PKR (B), Stat1 (C), or IFN-β (D) mRNA in each sample in quadruplicate. P values from a two-tailed t test assuming nonequal variance were computed for each mutant cell type compared to the corresponding WT cells for each infection and are indicated (*, P ≤ 0.005).

Article Snippet: The minor-groove binding probe and primer sets for each gene were part of an Applied Biosystems assay set as follows: Mm01207402_m1 for mouse TLR3, Mm00440966_m1 for mouse PKR, Mm01257286_m1 for mouse Stat1, and Mm00439546_s1 for mouse IFN-β.

Techniques: Virus, Infection, Isolation, Reverse Transcription, Quantitative RT-PCR, Two Tailed Test, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Five nucleotides found in RCTG motifs are essential for post-fertilization methylation imprinting of the H19 ICR in YAC transgenic mice

doi: 10.1093/nar/gkad516

Figure Lengend Snippet: Search for binding motifs for CD (= A) factor in the 118 bp fragment. (A) The 118 bp sequence and its subregions (open rectangles) used in the EMSA. Positions of (C)RCTG motifs are shown as horizontal thick arrows (I–V). (B) Sequences of fragments A–E and CD, as well as their mutant derivatives [A(Δ2)–E(Δ2) and CD(Δ3)]. The (C)RCTG motifs in each fragment are inverted in black-and-white, in which mutated bases are shown in black letters on a white background. (C–F) EMSA with nuclear extracts from P19 or ES cells, and probes A and B ( C ), C ( D ), E ( E ) and CD ( F ). Twenty-fold ( C ) or increasing amount (100–200–500–1000-fold in ( D ), 20–50–100–200-fold in ( E ) and 20–100-fold in ( F ) molar excess of oligos were used as competitors.

Article Snippet: Two sets of oligonucleotides were annealed and inserted at the Bbs I site of the pX330 (plasmid #42230; Addgene) ( ) to generate Cas9/sgRNA expression vectors.

Techniques: Binding Assay, Sequencing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Five nucleotides found in RCTG motifs are essential for post-fertilization methylation imprinting of the H19 ICR in YAC transgenic mice

doi: 10.1093/nar/gkad516

Figure Lengend Snippet: Search for binding motifs for CD (= A) factor in the fragment CD. (A) Sequences of fragment CD and its mutant derivatives. The (C)RCTG motifs in each fragment are inverted in black-and-white, in which the mutated bases are reverted. The binding affinity of each sequence to the CD (= A) factor, which was estimated by visual-examination of the EMSA results by three individuals, is indicated by ++ (strong), + (moderate) and – (weak) to the right of each sequence. (B, C) EMSA with nuclear extract from P19 cells and probe CD. Forty- and 200-fold molar excess of oligos were used as competitors. (D) Sequences of fragment CD and its scrambled mutants. Sequence composition ( i.e . the number of each nucleotides) is maintained even after shuffling the sequence. (E) EMSA with P19 cell nuclear extract and probe CD. Twenty- and eighty-fold molar excess of oligos were used as competitors. (F) Putative binding site for nuclear receptor-type transcription factors ( i.e . direct repeat sequence) is indicated by a pair of arrows. (G–I) EMSA with nuclear extracts from P19 or testis cells and probe F. One hundred-fold molar excess of oligos were used as competitors. Nucleotide sequences of ‘Epsi’ and ‘RARE’ oligos each carrying binding motifs for TR2/TR4 are as follows: Epsi, 5’-CTG AGGACA C AGGTCA GCCT TGACCA A TGACTT TTA-3’ and RARE, 5’-TTGCTG TGACCT C TGCCCT TCTAGCCTCT-3’ (only the sequence of one strand is shown and binding motifs are underlined). In Figure , the super-shift bands observed in the presence of antibodies against either TR2 or TR4 are indicated by an open triangle. In Figure , nuclear extracts were prepared from P19 cells transfected with siRNA duplexes against TR2, TR4, or both. The decrease in protein levels in these cells was verified by western blotting. NC; non-targeting control siRNA.

Article Snippet: Two sets of oligonucleotides were annealed and inserted at the Bbs I site of the pX330 (plasmid #42230; Addgene) ( ) to generate Cas9/sgRNA expression vectors.

Techniques: Binding Assay, Mutagenesis, Sequencing, Transfection, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Five nucleotides found in RCTG motifs are essential for post-fertilization methylation imprinting of the H19 ICR in YAC transgenic mice

doi: 10.1093/nar/gkad516

Figure Lengend Snippet: Ability of the CD factor to bind various DNA sequences. (A) Comparison between mouse 118 bp and rat 113 bp sequences. Identical nucleotides are denoted by vertical lines. The (C)RCTG motifs are inverted in black-and-white, while Zfp57-like motifs are shown in rectangles. Sequence portions used as probes in EMSA are indicated by horizontal lines. The Δ5 mutations are shown in lowercase letters above the mouse sequence. (B, E) EMSA with P19 cell nuclear extract and probe CD(WT). Twenty- and eighty-fold molar excess of oligos were used as competitors. (C) EMSA with P19 cell nuclear extract and probe F. Ten- and 40-fold molar excess of oligos were used as competitors. (D) RCTG-motif-containing sequences from the mouse H19 ICR, the repeat within the IG-DMR ( Dlk1–Dio3 gene locus), and the Sp4 repeat ( Rasgrf1 gene locus). The (C)RCTG consensus motifs are inverted in black and white, while Zfp57-binding consensus motifs are shown in red. Zfp57-binding consensus-like motifs (underlined) in the CD(WT) sequence were mutated to generate Zfp57-binding consensus motifs within the CD(Zfp57) sequence.

Article Snippet: Two sets of oligonucleotides were annealed and inserted at the Bbs I site of the pX330 (plasmid #42230; Addgene) ( ) to generate Cas9/sgRNA expression vectors.

Techniques: Comparison, Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Targeting parvalbumin promotes M2 macrophage polarization and energy expenditure in mice

doi: 10.1038/s41467-022-30757-y

Figure Lengend Snippet: a Parvalbumin protein expression in muscle and serum from WT and parvalbumin KO mice. b Representative M2 macrophage marker gene expression in scWAT and BAT of WT and parvalbumin KO mice. WT, n = 10; PVALB KO, n = 10. c Representative flow cytometry plots and ( d ) quantification demonstrate the numbers of M2 macrophages (CD206 + /CD11C − ) in the scWAT of WT and parvalbumin KO mice. WT, n = 13; PVALB KO, n = 13. e Male parvalbumin KO mice were intravenously injected with Ad-parvalbumin or Ad-GFP through the tail vein. Parvalbumin protein expression in the liver and serum were examined. f Representative M2 macrophage marker gene expression in scWAT and BAT of parvalbumin KO mice injected with Ad-GFP or Ad-parvalbumin. Adv-GFP, n = 8; adv-PVALB, n = 8. g Representative flow cytometry plots and ( h ) quantification demonstrate the numbers of M2 macrophages (CD206 + /CD11C − ) in the scWAT of parvalbumin KO mice injected with Ad-GFP and Ad-parvalbumin. Adv-GFP, n = 14; Ad-parvalbumin, n = 14. i Macrophage parvalbumin deficiency had no effect on M2 activation. Cd301, Cd206 , and Tgm2 mRNA expression in WT and parvalbumin KO BMDM treated with IL4 and parvalbumin (100 ng/ml) ( n = 3 per group). Data are represented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test ( b , d , f , h , and i ). Source data are provided as a Source Data file.

Article Snippet: Human PBMC were cultured in the presence of IL4 or MCSF for 24 h with or without recombinant human parvalbumin (15333-H08E, Sino Biological, China) and subjected to real-time PCR or immunoblot analysis.

Techniques: Expressing, Marker, Flow Cytometry, Injection, Activation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting parvalbumin promotes M2 macrophage polarization and energy expenditure in mice

doi: 10.1038/s41467-022-30757-y

Figure Lengend Snippet: a Representative immunoblots showing effects of parvalbumin (100 ng/ml) treatment on IL4-induced STAT6 phosphorylation at indicated time in BMDMs. b Representative immunoblots showing effects of parvalbumin (100 ng/ml) treatment on MCSF-induced mTORC2 downstream targets and ERK activation at indicated time in BMDMs. Representative immunoblots showing effects of varying concentrations of parvalbumin treatment on MCSF-induced mTORC2 downstream targets and ERK activation in BMDMs (concentrations of parvalbumin: 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml) ( c ) and human PBMCs (concentrations of parvalbumin:10 ng/ml, 100 ng/ml) ( d ). e examination of mTORC2 activity through in vitro kinase assay. f Co-localization of parvalbumin with transiently transfected CSF1R in COS7 cells. Scale bar, 20 μm. g Interaction between parvalbumin and immunoprecipitated GFP-tagged CSF1R. h Measurement of the binding affinity between parvalbumin and CSF1R proteins by SPR method. i Concentration-response curve of MCSF on CSF1R activation detected by ELK1-Luc in cells stably expressing CSF1R (MCSF EC50 = 0.36 ± 0.03 nM, n = 3 per group). j Concentration-response curve of parvalbumin on ELK1-Luc, showing that parvalbumin alone does not activate CSF1R ( n = 3 per group). k Concentration-response curve of parvalbumin on CSF1R activation in the presence of MCSF (parvalbumin IC50 = 3.26 ± 0.06 nM) ( n = 3 per group). l Concentration-response curve of parvalbumin on CSF1R activation in the presence of increasing concentrations of parvalbumin, showing that parvalbumin is a noncompetitive antagonist of CSF1R ( n = 3 per group). Data are represented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Human PBMC were cultured in the presence of IL4 or MCSF for 24 h with or without recombinant human parvalbumin (15333-H08E, Sino Biological, China) and subjected to real-time PCR or immunoblot analysis.

Techniques: Western Blot, Activation Assay, Activity Assay, In Vitro, Kinase Assay, Transfection, Immunoprecipitation, Binding Assay, Concentration Assay, Stable Transfection, Expressing