Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a – c Immunoblot analysis of FABP5 binding to ULCFA ( a ), SLCFA ( b ) or ULCFA with Grx1 treatment ( c ). Purified recombinant FABP5 protein was incubated with GSH plus H 2 O 2 or GSSG for 15 min, then mixed with Biotin-ULCFA or Biotin-SLCFA in the absence or presence of BMS309403 or linoleic acid or Grx1. FABP5 interacted with the Biotin-ULCFA or Biotin-SLCFA was captured using streptavidin magnetic beads. d Immunofluorescence staining of BMDMs after exposure to H 2 O 2 (200 μM) for 30 min (green, FABP5; blue, DAPI; scale bars, 10 μm). e Quantitative analysis of the ratio of nuclear/cytoplasmic fluorescence intensity in BMDMs as in ( d ), n = 10 in each group, P (ctr vs. ctr H2O2) = 0.0172, P (ctr H2O2 vs. Grx1 KO H2O2) <0.0001. f Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells. COS-7 cells were transfected with pXJ40-3xFlag vector or pXJ40-3xFlag-FABP5 followed by exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. g Immunoblots with antibodies against the indicated proteins in nuclear and cytoplasmic fractions from COS-7 cells co-transfected with pXJ40-3xFlag-FABP5 (or vector) and pcDNA3.1-3xFlag-Grx1 (or vector) for 24 h, then exposed to H 2 O 2 (200 μM) for 1 h. h Immunoblots against the indicated proteins of cytoplasmic and nuclear extracts from BMDMs exposed to H 2 O 2 (200 μM) for 1 h. i mRNA expression of Il1b , Il6 , and Tnfα in BMDMs from mice stimulated with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr vs. Ctr+LPS) = 0.0013, P ( Il1b , Ctr+LPS vs. Grx1 KO+LPS) = 0.0091, *** P < 0.0001. j mRNA levels of Il1b , Il6 , and Tnfα in BMDMs pre-treated with an FABP5 inhibitor (SBFI26, 100 μM) for 2 h and stimulated with LPS (100 ng/mL) for 4 h, as determined by qPCR, n = 3 in each group, *** P < 0.0001. k mRNA levels of Il1b , Il6 , and Tnfα in BMDMs after treated with SBFI26 (100 μM) for 2 h and stimulation with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Il16 , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Tnfα , Ctr+LPS vs. Ctr+LPS+inhibitor) = 0.0046. All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for (e, i-k). * P < 0.05, ** P < 0.01, *** P < 0.001, ns = no significance. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: Western Blot, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads, Immunofluorescence, Staining, Fluorescence, Transfection, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Model structure of FABP5 and its 6 cysteines, generated by Pymol and based on the crystal structure of Homo sapiens FABP5 (PDB ID,4LKT). b Mass spectra of a peptide from FABP5 including glutathionylated cysteine 120 and cysteine127. c Immunofluorescence staining (with FABP5) and confocal microscopy imaging of COS-7 cells with overexpression of FABP5 WT, C67S, C87S, C120S, and C127S after exposure to H 2 O 2 (200 μM) for 1 h (green, FABP5; blue, DAPI; scale bars, 10 μm). d Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells transfected with FABP5 WT or C127S after exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. e Model structure of FABP5 interaction with an FA (Fatty Acid, Linoleic Acid), generated by Pymol and based on the crystal structure of FABP5 (PDB ID, 4LKT). f Docking structure of FABP5 in complex with S-glutathionylated Cys127 interacting with an FA (Fatty Acid, Linoleic Acid). g Docking structure of dimer FABP5 (PDB ID, 4AZM, Homo sapiens ) in complex with reduced glutathione (GSH) on Cys127. h Co-IP for S-glutathionylation of FABP5 in COS-7 cells overexpressing pXJ40-3xFlag-FABP5 WT or C127S and exposed to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole-cell lysates confirm the expression of FABP5 and β-actin (DTT, negative control). i Immunoblot analysis of FABP5 fatty acid binding. Purified recombinant FABP5 WT or C127S protein (1 μg) was incubated with GSSG (1 mM) for 15 min, then mixed with Biotin-linoleic acid in the absence or presence of BMS309403 (5 mM) for 30 min. FABP5 that associated with the Biotin-linoleic acid was captured using streptavidin magnetic beads. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: Generated, Immunofluorescence, Staining, Confocal Microscopy, Imaging, Over Expression, Western Blot, Transfection, Co-Immunoprecipitation Assay, Expressing, Negative Control, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads

Journal: bioRxiv

Article Title: Spatiotemporal and single-cell atlases to dissect cell lineage differentiation and regional specific cell types in mouse ovary morphogenesis

doi: 10.1101/2023.07.21.549985

Figure Lengend Snippet: Molecular diversity and spatial distribution differences of luteal cells in mouse ovary. (A) UMAP visualization of marker genes for LLC and SLC. Color key represents the expression levels. LLC, large luteal cells; SLC, small luteal cells. (B) Visualization of LLC and SLC on 2-month mouse ovary based on integration of scRNA-seq cell type annotations with spatial transcriptomic data. (C) Immunohistochemistry staining of Onecut2 in mouse ovary. The scale bars represent 400 μm in low-magnification view and 60 μm in high-magnification view. (D) Immunohistochemistry staining of Onecut2 during ovulation. The scale bars represent 200 μm. (E) Spatial scatter pie plot representing the proportions of the LLC (left) and SLC (right) from the PD90 ovary reference atlas in the 2-month mouse ovary. (F) Heatmap showing the correlations among different cell clusters by Spearman analysis. (G) Pseudotime trajectory of granulosa cells, luteal cells and Theca cells analyzed by Monocle. (H) Spatial trajectory showing the differentiation of granulosa cells analyzed by stLearn. (I) Heatmap of regulon activity analyzed by SCENIC. (J) The outgoing signaling patterns among different cell clusters (left); The typical outgoing signaling pathway of SLC (right). (K) The incoming signaling patterns among different cell clusters (left); The typical incoming signaling pathway of SLC (right).

Article Snippet: The antibodies used were Onecut2 (Proteintech, 21916-1-AP).

Techniques: Marker, Expressing, Immunohistochemistry, Staining, Activity Assay

Journal: bioRxiv

Article Title: Spatiotemporal and single-cell atlases to dissect cell lineage differentiation and regional specific cell types in mouse ovary morphogenesis

doi: 10.1101/2023.07.21.549985

Figure Lengend Snippet: Identification of luteal cells and their specific spatial distributions. (A) Visualization of Theca cells, immune cells, mesenchyma cells and proliferate mesenchyma cells (pMesenchyma) on 2-month mouse ovary respectively based on integration of scRNA-seq cell type annotations with spatial transcriptomic data. (B) Visualization of large luteal cells (LLC), lymph associated endothelial cells (LEndo), Theca cells, immune cells, mesenchyma cells and proliferate mesenchyma cells (pMesenchyma) on another 2-month mouse ovary respectively based on integration of scRNA-seq cell type annotations with spatial transcriptomic data. (C) Venn diagram of top 200 differentially expressed genes in small luteal cells (SLC) and large luteal cells (LLC) demonstrates overlap between the gene populations in two subclusters of luteal cells. (D) The top GO terms enriched in the differentially expressed genes that unique to SLC (149), LLC (149) and shared genes (51) between LLC and SLC in . (E) The expression pattern of Onecut2 during ovary development. 3W, 3weeks; 3M, 3 months; 3M/PH, the ovary collected from 3-month mouse injected by pregnant mare serum and human chorionic gonadotropin; E11.5, embryonic day 11.5 ovary. (F) Spatial scatter pie plot representing the proportions of the LLC from the PD90 ovary reference atlas in the 2-month mouse ovary. (G) The spatial interactions among different cell clusters analyzed by SPOTlight. (H) Spatial trajectory showing the differentiation of granulosa cells in 2-month ovary section analyzed by stLearn. (I) The expression patterns of differentially expressed genes along trajectory. (J) Dynamic expression patterns of spatial trajectory associated ordering genes along SPATA trajectory.

Article Snippet: The antibodies used were Onecut2 (Proteintech, 21916-1-AP).

Techniques: Expressing, Injection

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a Kaplan–Meier survival analysis of progeny of the ENU-treated Th-MYCN mouse #1590, that were either wild-type for Runx1t1 (red, unsuppressed) or had inherited the Y534H Runx1t1 mutation (black, suppressed); n = 11 per genotype, p < 0.0001 (log-rank (Mantel–Cox) test). b Scatter plot showing time to tumor development in progeny of the ENU-treated mouse #1590 that were wild-type for Runx1t1 (red), or had inherited the Y534H mutation (black), n = 11 per genotype. c Multiple protein sequence alignment of the RUNX1T1 NHR4 domain across a range of organisms. The suppressed tumor phenotype resulted from the substitution of histidine (H) for a highly conserved tyrosine residue (Y), denoted by the asterisk (upper panel). A schematic model of wild-type and mutant Runx1t1 NHR4 zinc-finger motif domain folding (bottom panel). d Kaplan–Meier survival analysis for homozygous Th-MYCN mice either wild type ( Th-MYCN +/+ Runx1t1 +/+) or with heterozygous knock-out ( Th-MYCN +/+ Runx1t1 +/−) of Runx1t1 . Wild-type Runx1t1 mice (red) demonstrated almost complete tumor penetrance, while Runx1t1 heterozygous knock-out mice (black) almost entirely lacked the ability to form tumors ( n = 101 for wild-type and n = 163 for heterozygous knock-out, log-rank (Mantel–Cox) test p < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Mutagenesis, Sequencing, Residue, Knock-Out

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a The percentage neuroblast hyperplasia scored from homozygous Th-MYCN (+/+) mice or littermate mice lacking the MYCN transgene (−/−), with either wild-type (+/+) or heterozygous loss (+/−) of Runx1t1 . Scoring of N = 3–8 independent mice was performed for each genotype and timepoint. All data points were N = 3, except for +/+, +/+ week 1 and week 2 ( N = 4); +/+, +/− day 0 ( N = 8) and week 4 ( N = 4); −/−, +/− day 0 ( N = 6), week 1 ( N = 4) and week 4 ( N = 5). The graph is mean ± SEM. b Representative histology of RUNX1T1 staining in ganglia from mice homozygous for the Th-MYCN transgene, and either wild-type or heterozygous for Runx1t1 from day 0 and 4 weeks of age. Neuroblast hyperplasia is defined as ≥30 small round blue cells within a sympathetic ganglion . Photos were taken at 600X magnification, and the scale bars represent 20 microns. c βIII-tubulin staining of sympathetic ganglia isolated from Th-MYCN mice. The percent coverage of neurites was calculated and the area under the curve determined for each ganglia. N = 4 (+/+, +/−) or 5 (−/−, +/+ and +/+, +/+), Graphs are Mean ± SEM, * p = 0.0181, *** p = 0.0007. Two-tailed unpaired t -test. d Heatmap displaying gene clustering following RNA-Seq analysis of ganglia dissected from two-week-old Th-MYCN mice with either homozygous or heterozygous Runx1t1 , compared to fully developed murine neuroblastoma tumors. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Staining, Isolation, Two Tailed Test, RNA Sequencing

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a Runx1t1 and MYCN mRNA expression in liver, brain, and tumor samples from homozygous Th-MYCN mice with intact Runx1t1 relative to the control, Gusb . Means ± SEM ( n = 3) are indicated. Data were analysed using one-way-ANOVA and corrected for the multiple comparison using the Tukey test. *** p = 0.0042, ns p = 0.1385 for Runx1t1 ; *** p = 0.0006 for MYCN . Three independent mice were used for each tissue. b Western blot for RUNX1T1 and MYCN in liver, brain, and tumor from homozygous Th-MYCN mice; n = 3 independent mice. c RUNX1T1 mRNA expression relative to the control GUSB in MYCN -non-amplified (non-AMP) and amplified (AMP) neuroblastoma cell lines. Means ± SEM ( n = 5 data points representing five different cell lines in each group, and the value of each data point is the mean of three biological repeats) are indicated, two-tailed Mann–Whitney test, P = 0.3095. d Western blot for RUNX1T1 and MYCN across a panel of MYCN non-amplified (non-AMP) and MYCN amplified (AMP) neuroblastoma cell lines. This experiment has been repeated once with similar results. e Scatter plot of RUNX1T1 mRNA expression in MYCN non-amplified (non-AMP) and amplified (AMP) tumors from a publicly available dataset (SEQC) of tumor samples from the neuroblastoma R2 database, n = 410 non-AMP and n = 92 AMP samples; two-tailed unpaired t -test, **** p < 0.0001. f A tissue microarray (TMA) of human neuroblastoma tumor samples ( n = 66) was stained with antibodies to either MYCN or RUNX1T1. Photos from three representative MYCN -non-amplified samples and three MYCN -amplified samples are shown. Numbers in panels indicate staining intensity. Photos were taken at 600× magnification, and the scale bars represent 20 microns. H&E, hematoxylin and eosin. g RUNX1T1 staining intensity was scored for all samples in the TMA. Neuroblastoma samples were split into non-amplified ( n = 52) and amplified ( n = 14) and compared to benign diseases of ganglioneuroblastoma (GNB) ( n = 5) and ganglioneuroma (GN) ( n = 12). Violin plot describes distribution of intensity, width describes frequency of score in tumors. The thick line represents the median, thin line represents the 25th and 75th percentiles. The bounds of the box show the range of scores. Unpaired t -test, two-tailed * p = 0.0403; ** p = 0.0064; *** p = 0.0001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Expressing, Control, Comparison, Western Blot, Amplification, Two Tailed Test, MANN-WHITNEY, Microarray, Staining

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a Western blot of SH-EP TET-21/N cells after 72 h of treatment with 1 µg/mL doxycycline (left panel). Quantitation from three independent experiments demonstrated significantly decreased MYCN (**** p = 0.0001) and RUNX1T1 expression (*** p < 0.0001) (right panel). Values represent means from three independent experiments ±SD, two-tailed unpaired t -test. b MYCN and RUNX1T1 mRNA expression after 72 h doxycycline treatment, showing significant downregulation of MYCN (*** p = 0.0009) but no significant change in RUNX1T1 ( p = 0.9559). Values represent means from three independent experiments ±SD, two-tailed unpaired t -test. c Western blot of SH-EP neuroblastoma cells overexpressing MYCN and empty vector (EV) control (left panel). Quantitation of western blot, from three independent experiments (right panel) demonstrated significantly increased RUNX1T1 expression (* p = 0.0339). Values represent means from three independent experiments ±SD, two-tailed unpaired t -test. d RUNX1T1 and MYCN mRNA expression in SH-EP cells overexpressing MYCN, relative to the control gene ( GUSB ). Values represent means from three independent experiments ±SD. Two-tailed unpaired t -test, **** p < 0.0001; ns: not significant ( p = 0.5679). e Cyclohexamide chase e xperiment showing RUNX1T1 protein stability in three neuroblastoma cell lines (BE(2)-C, KELLY, SH-EP) over a 12 h time course. Values represent means from three independent experiments ±SD. f Luciferase activity following transfection of murine Runx1t1 5’UTR into MYCN overexpressing or EV SH-EP cells. Values represent means from three independent experiments ±SD. Groups were analysed using two-way ANOVA and Bonferroni multiple comparison. *** p = 0.0007; ns not significant ( p = 0.9335). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Western Blot, Quantitation Assay, Expressing, Two Tailed Test, Plasmid Preparation, Control, Luciferase, Activity Assay, Transfection, Comparison

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a, b RUNX1T1 knock down after 72hrs of doxycycline (1 µg/mL) treatment in KELLY and BE(2)-C cells respectively, with two independent shRNA constructs. Quantitation from four independent experiments demonstrated significantly decreased RUNX1T1 levels following shRNA-mediated knockdown. Values represent means from three independent experiments ±SD. Ordinary one-way ANOVA with Tukey’s multiple comparisons test; KELLY sh#1 **** p < 0.0001, sh #2 ** p = 0.0018, ns p = 0. 6962; BE(2)-C sh #1 *** p = 0.0006, sh #2 * p = 0.0295, ns p = 0.8753. c , d Colony formation after doxycycline-induced knockdown of RUNX1T1 in KELLY and BE(2)-C cells respectively, with two independent shRNA constructs. Colonies are represented as a percentage relative to the untreated control in each experiment. Values represent means from three independent experiments ±SD. RUNX1T1 shRNA-mediated knockdown resulted in significantly decreased colony numbers compared to controls. Ordinary 1-way ANOVA with Tukey’s multiple comparisons test; KELLY sh #1 **** p < 0.0001, sh #2 **** p < 0.0001, ns p = 0. 3589; BE(2)-C sh #1 *** p = 0.0005, sh #2 **** p < 0.0001, ns p = 0.8549. e , f Kaplan–Meier survival analysis of NSG mice xenografted with doxycycline-inducible RUNX1T1 shRNA#1 in KELLY and BE(2)-C cells, respectively. Mice were split into two groups ( n = 10 mice per group for KELLY and n = 6 mice per group for BE(2)-C) and fed doxycycline or control food once a 50 mm 3 tumor was measurable. P values were determined using the Log-Rank (Mantel–Cox) test, p < 0.0001 for KELLY and p = 0.0035 for BE(2)-C. Growth curves (lower panels) plot size over time, post-doxycycline treatment. Graphs are Mean ± SEM. g Representative images of KELLY cells at 7 days and endpoint ± doxycycline. Tumor samples were stained with H&E or immunohistochemically for RUNX1T1 and Ki67. Photos were taken at 600× magnification, and the scale bars represent 20 microns. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Knockdown, shRNA, Construct, Quantitation Assay, Control, Staining

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a One-dimensional 1 H NMR spectra of the Runx1t1 MYND domain showed that the wild-type spectrum (top) has sharp peaks and good dispersion whereas the spectrum of the Y534H mutant (bottom) indicates a protein not well-ordered and/or aggregated—consistent with the mutation disrupting proper folding of the domain. b Co-IP between RUNX1T1_WT/YH-3xFLAG and LSD1-HA (top left), CoREST3-HA (top right), HDAC3-HA (middle left), HDAC1-HA (middle right), HDAC2-HA (bottom left), and HAND2-HA (bottom right). HEK-293T cells were transiently co-transfected with pCMV14 RUNX1T1_WT- or Y534H-3xFLAG and HA-tagged constructs. Nuclear fractions were immunoprecipitated with anti-FLAG antibody and immunoblotted with both anti-FLAG antibody (IP) and anti-HA (Co-IP). An experiment was performed twice. The input sample represents 5% of the non-immunoprecipitated sample and the control sample (EV) resulted from the co-transfection with the HA-tagged construct and the pCMV14 empty vector. c Summary of the Co-IP results. d Binding curves and calculated affinities for peptides RCOR3_1-3 and NCOR2 at pH 6 and RCOR3_3 and NCOR2 at pH 7.5. Binding curves were derived by tracking the combined 1 H and 15 N Chemical Shift Perturbations of three signals in the MYND domain 15 N-HSQC following titration of each peptide in increments of 0.5 molar equivalents into samples of 15 N-HNR4 MYND, each of which was fitted to a simple 1:1 binding model. The affinities reported for each peptide are the average (±SD) of three sets of measurements. We estimate the uncertainty for the KD’s to be ~25%. The affinity of NCOR2 at pH 7.5 was calculated by ref. and is listed for comparison. e Schema of CoREST3/RCOR3 deletion mutants (left). HEK-293T cells were transiently co-transfected with pCMV14 RUNX1T1_WT-3xFLAG and HA-tagged CoREST3 constructs (right, n = 1 experiment). Nuclear fractions were immunoprecipitated with anti-FLAG antibody and samples immunoblotted and probed with anti-FLAG antibody (IP, top boxes), or anti-HA (Co-IP, bottom boxes). Input represents 5% of non-immunoprecipitated sample; −/+ indicates co-transfection of pCMV14 EV and HA-CoREST3 constructs (−) or co-transfection of pCMV14 RUNX1T1_WT-3xFLAG and HA-CoREST3 constructs (+). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Dispersion, Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Construct, Immunoprecipitation, Control, Cotransfection, Plasmid Preparation, Binding Assay, Derivative Assay, Titration, Comparison

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a ChIP-seq analysis of RUNX1T1 binding across three replicates showing the number of peaks and their distance in base-pairs (bp) to the transcription start site (TSS). b Proportion of the peaks occurring at different binding site locations from the RUNX1T1 three ChIP-seq replicates. RUNX1T1 binding occurring at either a promoter, gene body, distal intergenic or downstream in relation to a gene (left), binding in an exon, intron or intergenic (middle), and binding within exons at either a coding region (CDS), 5’ untranslated region (UTR) or 3’ UTR (right). c Motif discovery using homer analysis showing significantly enriched motifs from HAND2, PHOX2A, and TCF4. d UpSet plot of the intersection of ChIP-seq peaks of RUNX1T1, HAND2, LSD1, and RCOR3. The number of peaks identified are indicated for each gene and the intersection of gene peaks. RUNX1T1 peaks that co-localized with HAND2, LSD1, and CoREST3 are shown by the blue bar. e GREAT analysis (using Binomial test) showing enriched gene ontology (GO) biological process of the common peaks between RUNX1T1, HAND2, LSD1, and RCOR3. f Number of active, poised, and primed enhancer sites where RUNX1T1 binds in the presence (wildtype; WT) and absence of RUNX1T1 (knock out; KO).

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: ChIP-sequencing, Binding Assay, Knock-Out

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a Gene set enrichment analysis (GSEA) for Hallmark gene sets performed on RNAseq data obtained following RUNX1T1 shRNA knockdown in KELLY cells. A bubble plot is shown with the size of the bubble representing the significance based on the nominal p value. NES: normalized enrichment score showing the strength and enrichment direction. The column labeled ganglia represents the same analysis performed on ganglia (Th-MYCN +/+ ; Runx1t1 +/− ) at 2 weeks versus tumor obtained from Th-MYCN +/+ ; Runx1t1 +/+ mice. b GSEA plot showing Hallmark MYC_target_V1 genes following RUNX1T1 shRNA downregulation in KELLY cells. c GSEA showed significant enrichment of PRC2 target genes (BenPorath_PRC2_targets) following RUNX1T1 shRNA downregulation in KELLY cells. d RUNX1T1 shRNA downregulation in aRMS cell lines (Rh41 and Rh3) significantly decreased cell proliferation, n = 3 independent experiments; graph is mean ± SEM, two-way ANOVA with Dunnett’s multiple comparison. Rh41 sh #1 ± Doxy 6d *** p = 0.0003, 7d and 8d **** p < 0.0001; Rh3 sh #1 ± Doxy 6d * p = 0.0149, 7d and 8d **** p < 0.0001. The immunoblots show decreased RUNX1T1 levels following shRNA-mediated knockdown. Each experiment has been repeated three times with similar results. e RUNX1T1 shRNA downr e gulation in SCLC cell lines (DMS-273 and DMS-53) significantly decreased clonogenic capacity, n = 3 independent experiments, graph is mean ± SEM, unpaired two-tailed t -test. DMS-273 EV ns p = 0.6682, sh#1 * p = 0.0127, sh#2 * p = 0.0465; DMS-53 EV ns p = 0.2210, sh#1 * p = 0.0325, sh#2 ** p = 0.0075. The immunoblots show decreased RUNX1T1 levels following shRNA-mediated knockdown. Each experiment has been repeated three times with similar results. f Proposed mechanism of action of RUNX1T1 in MYCN -amplified neuroblastoma. High levels of MYCN resulting from gene amplification drive increased protein translation which includes increased RUNX1T1 protein levels. High-level RUNX1T1 expression is necessary to maintain an ESC-like phenotype as well as generate enhancer-mediated repression of HAND2 targets genes otherwise required for neuronal differentiation. Loss of RUNX1T1 allows upregulation of HAND2-driven pro-differentiation genes while at the same time inhibiting MYCN activity by decreasing the level of the obligate MYCN dimerization partner MAX. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: shRNA, Knockdown, Labeling, Comparison, Western Blot, Two Tailed Test, Amplification, Expressing, Activity Assay