Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Increased cortical expression of two synaptogenic thrombospondins in human brain evolution.

doi: 10.1093/cercor/bhl140

Figure Lengend Snippet: Figure 1. THBS4 and THBS2 expression levels in human (Hs) and chimpanzee brain (Pt) from oligonucleotide microarrays. Graphs show the average hybridization signal intensity and standard error in different brain regions of each species. The brain regions are FCx, frontal cortex (FP, middle frontal gyrus, and Brodmann area 9); TCx, temporal cortex (Broca’s area, TP, aIT cortex, and superior temporal gyrus); VCx, primary visual cortex; ACCx, anterior cingulate cortex; Cau, caudate nucleus; Cb, cerebellar vermis. The number of individuals analyzed was 9 humans and 5 chimpanzees for FCx, 6 humans and 6 chimpanzees for TCx, and 3 humans and 3 chimpanzees for the other brain regions. The average hybridization signal resulting from combining together all the forebrain regions (FCx, TCx, VCx, ACCx, and Cau) is represented by dashed and dotted lines in humans and chimpanzees, respectively. Asterisks indicate the results of the Mann--Whitney test for the comparison of THBS4 and THBS2 levels for each region between humans and chimpanzees. *P \ 0.05; **P \ 0.01.

Article Snippet: Based on trial studies, we used the following dilutions of the same polyclonal antibodies against THBS4 used for Western blotting: R&D Systems antibody, 1:20 and 1:25; Santa Cruz Biotechnology antibody, 1:40; and Lawler 1259 antibody, 1:500.

Techniques: Expressing, Hybridization, MANN-WHITNEY, Comparison

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Increased cortical expression of two synaptogenic thrombospondins in human brain evolution.

doi: 10.1093/cercor/bhl140

Figure Lengend Snippet: Figure 3. THBS4 and THBS2 expression levels in nonbrain tissues from humans and nonhuman primates. (A) Oligonucleotide microarray results for THBS4 and THBS2 in nonbrain tissues of humans and chimpanzees. Graphs show the average hybridization signal in different tissues of 6 humans (Hs) and 5 chimpanzees (Pt), including brain frontal cortex for comparison. (B) Real-time RT-PCR quantification of THBS4 and THBS2 expression levels in heart from different primate species. The average number of copies of thrombospondin (THBS) mRNA for 1000 b-actin (ACTB) mRNA copies in humans, chimpanzees, and rhesus macaques is represented on the y axis. Error bars represent standard errors. The results of the Mann--Whitney test comparing THBS4 and THBS2 expression levels between humans and nonhuman primates are represented by asterisks. *P \ 0.05; **P \ 0.01.

Article Snippet: Based on trial studies, we used the following dilutions of the same polyclonal antibodies against THBS4 used for Western blotting: R&D Systems antibody, 1:20 and 1:25; Santa Cruz Biotechnology antibody, 1:40; and Lawler 1259 antibody, 1:500.

Techniques: Expressing, Microarray, Hybridization, Comparison, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Increased cortical expression of two synaptogenic thrombospondins in human brain evolution.

doi: 10.1093/cercor/bhl140

Figure Lengend Snippet: Figure 4. Quantification of THBS4 and THBS2 protein levels in primate frontal cortex by Western blot analysis. (A) Western blot results for THBS4 (103.5 kDa), THBS2 (129.0 kDa), and b-tubulin (TUBB) (49.8 kDa) using FP samples of 3 humans (Hs), 3 chimpanzees (Pt), and 3 rhesus macaques (Mm). For each protein, one representative blot is shown on top and the average band intensity from the 3 different blots quantified is shown below. In each blot, band intensities were normalized to those of one human case (Hs2). (B) Average THBS4 and THBS2 protein levels in the FP of humans, chimpanzees, and rhesus macaques. The y axis corresponds to the average band intensities of the 3 individuals of each species relative to the human value, after normalization by TUBB levels to control for protein loading differences. Error bars represent standard errors. Asterisks indicate the results of the Mann--Whitney test for the comparison of thrombospondin levels between humans and nonhuman primates. *P \ 0.05.

Article Snippet: Based on trial studies, we used the following dilutions of the same polyclonal antibodies against THBS4 used for Western blotting: R&D Systems antibody, 1:20 and 1:25; Santa Cruz Biotechnology antibody, 1:40; and Lawler 1259 antibody, 1:500.

Techniques: Western Blot, Control, MANN-WHITNEY, Comparison

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Increased cortical expression of two synaptogenic thrombospondins in human brain evolution.

doi: 10.1093/cercor/bhl140

Figure Lengend Snippet: Figure 5. Histological localization of THBS4 mRNA in primate frontal cortex by in situ hybridization. (A--C) Low-magnification photomicrographs of the frontal polar cortex of a human (A), a chimpanzee (B), and a rhesus macaque (C), showing the hybridization of the THBS4 antisense probe in unfixed, snap-frozen sections. (D--E) High- magnification photomicrographs of fixed tissue sections showing numerous pyramidal cells labeled for THBS4 mRNA in cortical layer 3 of a human (D) and a chimpanzee (E). Scale bars: (A--C) 250 lm; (D--E) 50 lm.

Article Snippet: Based on trial studies, we used the following dilutions of the same polyclonal antibodies against THBS4 used for Western blotting: R&D Systems antibody, 1:20 and 1:25; Santa Cruz Biotechnology antibody, 1:40; and Lawler 1259 antibody, 1:500.

Techniques: In Situ Hybridization, Hybridization, Labeling

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Increased cortical expression of two synaptogenic thrombospondins in human brain evolution.

doi: 10.1093/cercor/bhl140

Figure Lengend Snippet: Figure 6. THBS4 mRNA expression in various cell types of human frontal cortex. (A-- B) Gray and white matter sections double labeled by in situ hybridization for THBS4 mRNA (blue staining) and by immunocytochemistry for the astrocyte-specific marker GFAP (brown staining). (A) In gray matter, arrowheads denote THBS4-expressing astrocytes in layer 1 and in deeper layers of the cortex. Not all GFAP-immunoreactive cells were clearly labeled with the THBS4 antisense probe, however. The cells in layers 2 and 3 exhibiting blue label only were probably neurons. (B) In the white matter (WM), a large population of small cells labeled strongly for THBS4 mRNA but did not stain for GFAP (arrowheads). Based on their number and size, these were probably oligodendrocytes. (C) Endothelial cells in the wall of a cerebral blood vessel labeled by in situ hybridization for THBS4 mRNA are indicated by arrowheads. The red coloration of the vessel is from blood. Scale bars: (A--B) 50 lm; (C) 10 lm.

Article Snippet: Based on trial studies, we used the following dilutions of the same polyclonal antibodies against THBS4 used for Western blotting: R&D Systems antibody, 1:20 and 1:25; Santa Cruz Biotechnology antibody, 1:40; and Lawler 1259 antibody, 1:500.

Techniques: Expressing, Labeling, In Situ Hybridization, Staining, Immunocytochemistry, Marker

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Increased cortical expression of two synaptogenic thrombospondins in human brain evolution.

doi: 10.1093/cercor/bhl140

Figure Lengend Snippet: Figure 7. Localization of THBS4 protein by immunohistochemistry in the frontal polar cortex of humans, chimpanzees, and macaques. (A--C) High-magnification photo- micrographs with differential-interference contrast optics of 5-lm-thick, paraffin- embedded sections through the upper part of cortical layer 3 of a human (A), a chimpanzee (B), and a rhesus macaque (C). (D--F) Representative low-magnification photomicrographs from 50-lm-thick fixed sections through cortical layers 1--6 of a human (D), a chimpanzee (E), and a rhesus macaque (F). In all species, THBS4 antibodies labeled numerous cell bodies, including many pyramidal cells in layers 2--6. Humans, however, were distinguished by dense labeling of the neuropil surrounding cell bodies. Scale bars: (A--C) 50 lm; (D--F) 250 lm.

Article Snippet: Based on trial studies, we used the following dilutions of the same polyclonal antibodies against THBS4 used for Western blotting: R&D Systems antibody, 1:20 and 1:25; Santa Cruz Biotechnology antibody, 1:40; and Lawler 1259 antibody, 1:500.

Techniques: Immunohistochemistry, Labeling

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Increased cortical expression of two synaptogenic thrombospondins in human brain evolution.

doi: 10.1093/cercor/bhl140

Figure Lengend Snippet: Figure 8. THBS4 immunostaining of Ab-containing plaques in gray matter from human cortex. (A) Section of frontal cortex from an Alzheimer’s case labeled with anti- THBS4 antibody and DAB (red-brown staining), revealing numerous plaque-like accumulations in the upper cortical layers, several of which are denoted by arrowheads. (B) A neighboring section stained for Ab peptide with 4G8 antibody and Vector Nickel (dark staining), revealing numerous well-defined Ab-containing plaques. (C) Higher-magnification photomicrograph of a section from the same case double-labeled for THBS4 with DAB (red-brown staining) and for Ab protein with Vector Nickel (dark staining). The sequential double-staining protocol yielded a mosaic of small red-brown and dark purple territories within the plaques. Scale bars: (A--B) 100 lm; (C) 50 lm.

Article Snippet: Based on trial studies, we used the following dilutions of the same polyclonal antibodies against THBS4 used for Western blotting: R&D Systems antibody, 1:20 and 1:25; Santa Cruz Biotechnology antibody, 1:40; and Lawler 1259 antibody, 1:500.

Techniques: Immunostaining, Labeling, Staining, Plasmid Preparation, Double Staining

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) Anonymized patient IDs and their color code used for data display. B) Schematic of key processing steps of snRNA-seq and snATAC-seq library generation. Nuclei were released from cryopreserved mammary tissue through mechanical homogenization, sorted to enrich for singlets and directly used as input for 10X Genomics library preparation. C) Schematic design of 4×4 tissue microarrays used in CODEX experiments, colors indicating sample arrangement. We used 7 cis-female and 9 trans-male samples, using 8 (2 mm) tissue regions from left and right breast of each sample. We block-randomized the samples so that each TMA contains both cis-female and trans-male samples. D) Heatmap shows the markers we used to identify cell types in snRNA and snATAC data. Values indicate scaled averages of RNA-expression in snRNA-seq (left) and gene activity scores in snATAC-seq data sets (right). E) Number of cell type specific chromatin accessibility peaks within each cell type. F) Relative proportion of each cell type within snRNA-seq (left) and snATAC-seq data (right). G) Scatterplot shows the fraction of each cell type within each of the 18 samples in snRNA-seq data (horizontal axis) and snATAC-seq data (vertical axis). The dashed diagonal represents the identity line (p-value = 1.85 x 10 -28 ). H) Heatmap shows the scaled average staining intensities corresponding to each of the cell class markers within each group of cells in the CODEX tissue microarray dataset. I) UMAP shows identity of cells in CODEX tissue microarray dataset determined through clustering based on staining intensities of cell class markers. Barplots show the proportion of each cell class within each of the 16 samples we used for CODEX tissue microarray. J) Boxplot shows age of the trans-male samples (orange), cis-female pre-menopausal samples (light purple), and cis-female post-menopausal samples (dark purple).

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: Homogenization, Blocking Assay, RNA Expression, Activity Assay, Staining, Microarray

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) snRNA-seq UMAP showing detected subclusters of hormone receptor expressing luminal cells (luminal-HR + ), with RNA-velocity streams overlayed and gender identity shown by sample (right panels). B) RNA-velocity pseudotime ordering of trans-male and cis-female luminal-HR + cells. Time 0 (T0) in the center and respective endpoints of cis-female and trans-male lineages (T1) at the outer maxima. Annotation bars show gender identity and subcluster assignment of each cell. Rows are annotated with highly differentially expressed genes or subcluster markers. C) Left panel overlays hormone receptor RNA expression on UMAP from (a). Right panel shows boxplots of hormone receptor staining intensities averaged across luminal-HR + cells in CODEX microarray regions of cis-female (purple) and trans-male (orange) tissues. Dot colors indicate from which TMA the shown regions originate. (p-value, Wilcoxon: PGR = 0.00041) D) Violin plot (left) showing per cell chromVAR motif enrichment z-scores for AR (CisBP M03389_2.00) from luminal-HR + cells in snATAC-seq (p-value, Wilcoxon < 2.2 x 10 -16 ). Boxplot (right) showing average nuclear to cytoplasmic staining ratio for AR in luminal-HR + cells from each TMA region (p-value, Wilcoxon = 0.00021). Dot colors indicate from which TMA the shown regions originate. Purple corresponds to cis-female and orange corresponds to trans-male data. E) RYR2 chromatin accessibility (top) for cis-female (purple) and trans-male (orange) luminal-HR + cells, with highlighted motif binding sites of AR, FOXA1 and CTCF. The RYR2 gene body (light-green) is shown with promoter (arrow) and exon boundaries (dark-green). Also shown (center) is chromatin accessibility data for the same genomic region in tissues with varying RYR2 expression (right ventricle, left ventricle and pancreas, prostate, breast) and Hi-C data (bottom) comparing three-dimensional chromatin structure of the same region in PANC-1 (pancreas) and MCF-10A (breast) cell lines. F) AR motif binding sites (red markers) across open chromatin regions around the CUX2 locus of trans-male (orange) and cis-female (purple) luminal-HR + cells. CUX2 gene body (light-green), exon boundaries (dark-green) and promoter (arrow) are shown below. G) Per-sample average RNA (left, adjusted p-value, MAST < 2.2 x 10 -16 ) and per-region average staining intensity (right, p-value, Wilcoxon = 0.027) of CUX2 in cis-female (purple) and trans-male (orange) tissues. Dot colors indicate from which TMA the shown regions originate. H) Effect sizes of CUX2 sex bias in GTEx tissues shown as a function of median AR expression (vertical axis and dot-size). Positive and negative values indicate female and male bias, respectively. I) Chromatin accessibility (top) around the PGR locus in trans-male (orange) and cis-female (purple) luminal-HR + and luminal-HR − cells. The PGR gene body (light-green) is shown with promoter (arrow) and exon boundaries (dark-green). Bottom left shows a magnified view of an AR motif containing peak (location highlighted in gray above). On the bottom right are the importance levels of transcription factors that co-bind with AR and determine the directionality of the transcriptional change in the target (as identified through fitting a random forest to classify upregulated or downregulated AR-bound genes using the accessibility of the peaks of each gene at the occurrences of motifs). Binding sites of these transcription factors are visualized with colored markers in the genomic region above. J) Top left panel shows footprint of AR (red), JUN (green), and ESR1 (purple) in cis-female (left) and trans-male (right) luminal-HR + cells. Bottom left shows the average log2FC of chromatin accessibility in peaks containing only the ESR1 motif (orange), ESR1 and JUN motifs (purple), or no ESR1 motif (gray). Bottom right shows the fraction of JUN motif overlapping peaks among all luminal-HR + peaks (left), cis-female–specific luminal-HR + peaks (middle), or trans-male–specific luminal-HR + peaks (right) containing both ESR1 and JUN motif (purple) or only ESR1 motif (orange). Top right shows the fraction of peaks with both ESR1 and JUN motifs which had in vitro ChIP-seq evidence for binding of both JUN and ESR1 (purple).

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: Expressing, RNA Expression, Staining, Microarray, Binding Assay, Hi-C, In Vitro, ChIP-sequencing

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) Representative CODEX microarray images, showing hormone receptor staining (AR = red, ESR1 = blue, PGR = purple) among luminal epithelial (KRT8 = green) cells in cis-female (left) and trans-male (right) breast tissues. Both tissue regions are part of the same TMA. B) Heatmap shows average module scores of biological pathways with differential enrichment among the 6 subclusters of luminal-HR + cells. The top bar plot indicates the fraction of each subcluster among either trans-male (orange) or cis-female (purple) cells. C) Origin probability, as calculated by CellRank, among luminal-HR + cells overlaid in the corresponding UMAP from . Location of cells belonging to subcluster lup 4 is highlighted in red. D) Diffusion map of trans-male (top) and cis-female (bottom) luminal-HR + cells, generated with destiny. Colors represent subclusters from and subcluster lup 4 is labeled. E) Motif footprints for Androgen Receptor (AR, AR-CisBP M03389_2.00) among trans-male (orange) and cis-female (purple) luminal-HR + cells. Top panel shows the transposase bias-corrected signal and the bottom panel shows the transposase bias. F) Ratio of nuclear to cytoplasmic staining signal of hormone receptors ESR1 and PGR in cis-female (purple) and trans-male (orange) tissues on CODEX microarrays. Dot colors indicate from which TMA the shown regions originate. G) Right panel shows the enrichment of motifs among the peaks of trans-male (orange) and cis-female (purple) luminal-HR + cells. Left panel shows the fraction of the peaks of the corresponding cells which overlap with the motif. H) Average RYR2 staining intensity per tissue region of luminal-HR + cells in cis-female (purple) and trans-male (orange) tissue of CODEX microarray data. Dot colors indicate the origin TMA of shown regions (p-value, Wilcoxon = 0.024). I) KEGG Insulin secretion pathway annotated with average log 2 fold change expression of the involved genes. Higher values indicate a higher expression in trans-male luminal-HR + cells.

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: Microarray, Staining, Diffusion-based Assay, Generated, Labeling, Expressing

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) Gene expression of CUX2 transcription factor among the top 10 CUX2 -expressing tissues of the GTEx dataset. The horizontal axis shows median transcripts per million reads (TPM) among the different samples of each tissue. B) The heatmap shows the average sex-bias (as provided by GTEx) of upregulated (trans-male–specific) and downregulated (cis-female–specific) genes (|log 2 FC| = 0.5) of luminal-HR + cells among the cis-male or cis-female samples of each tissue. The figure shows the tissues with data in both cis-male and cis-female samples. Ordering of tissues represents their median AR gene expression. C) Receiver-operating characteristic curve (top) and precision-recall curve (bottom) assess the performance of the random forest on the held-out set of peaks. D) Average motif distance and total number of co-occurrences between AR and other transcription factor motifs in open chromatin regions of luminal-HR + cells. Purple horizontal dashed line indicates the median average motif distance. E) The heatmap shows the effect size of linear models which predict a transcription factor motif (vertical axis) z-score activity using the gene expression of the transcription factor (horizontal axis). F) Boxplots show per-region average BATF staining intensity in CODEX microarray data of cis-female (purple) and trans-male (orange) tissues. Dot colors indicate TMA origin of the shown regions. (p-value, Wilcoxon = 0.00014) G) Selected transcription factor binding sites (colored markers) in open chromatin regions around EREG and AREG in luminal-HR + cells. Transcription factors were selected based on differential expression and motif enrichment. The genomic window shows chromatin accessibility for trans-male (orange) and cis-female (purple) cells. Gene bodies of EREG and AREG (light-green) are shown with exon boundaries (dark green) and promoter location (arrow). H) Average RNA expression of AREG , EREG , and their receptor EGFR among luminal-HR + , luminal-HR − , and basal epithelial cells of cis-female (purple) and trans-male (orange) samples (adjusted p-values, MAST: AREG in luminal-HR + < 2.2 x 10 -16 , EREG in luminal-HR + < 2.2 x 10 -16 , EGFR in luminal-HR + = 4.37 x 10 -107 , luminal-HR - = 2.94 x 10 -205 , basal = 8.18 x 10 -117 ) I) Microscopic images from CODEX microarray data showing AREG staining (turquoise) and luminal-HR + cells (AR = red) in cis-female (left) and trans-male (right) tissues, each with arrows and zoomed-in panels to highlight AREG distribution around luminal-HR + cells. Both tissue regions are part of the same TMA. J) Left panel boxplots show per-sample average expression of ADAM17 among basal, luminal-HR − , and luminal-HR + cells of cis-female (purple) and trans-male (orange) (adjusted p-values, MAST: luminal-HR - < 2.2 x 10 -16 , basal = 1.32 x 10 -265 ). K) Boxplots show per-region average EGFR staining intensity of epithelial cell types in CODEX microarray data of cis-female (purple) and trans-male (orange) tissues. Dot colors indicate TMA origin of the shown regions. (p-value, Wilcoxon: luminal-HR + = 0.0069, luminal = 0.083, basal = 0.26)

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: Expressing, Activity Assay, Staining, Microarray, Binding Assay, RNA Expression

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) Heatmap showing the log 2 fold change in RNA expression of PI3K receptors (taken from “KEGG: PI3K-Akt signaling pathway”) among the 10 cell types of the breast. The diameter of the circle indicates the fraction of cis-female cells of the cell type expressing the receptor. INS indicates circulating insulin secreted in the pancreas. B) Heatmap showing the log 2 fold change in RNA expression of the downstream transcription factors of the “KEGG: PI3K-Akt signaling pathway” among the 10 cell types of the breast. C) Pathway enrichment of tissue wide upregulated genes (> 7 cell types) that also have an NR4A1 motif in an enhancer (> 4 cell types). Horizontal axis shows the odds ratio (Fisher’s exact test) comparing frequency of selected genes in the pathway versus background and vertical axis shows –log 10 p-value of Fisher’s exact test. FDR < 0.05 = red, p-value < 0.05 = yellow, n.s. = gray. (PID = Pathway Interaction Database, REAC = Reactome, WP = WikiPathways, KEGG: Kyoto Encyclopedia of Genes and Genomes). D) Module scores for the WikiPathways “WP: insulin signaling pathway” in all cell types, split by cis-female (purple) and trans-male (orange). (p-value in adipocytes, Wilcoxon = 7.59 x 10 -4 ). E) Representative images of computational segmentation of lipid vacuoles (left, see methods), and resulting average area of adipocyte vacuoles per IHC scan region (p-value Wilcoxon = 0.00059). F) Boxplot shows the sample averages of AZGP1 RNA-expression in each cell type in trans-male (orange) and cis-female (purple) samples (adjusted p-values, MAST: adipocyte = 5.95 x 10 -12 , basal = 2.42 x 10 -70 , blood EC = 8.67 x 10 -83 , fibroblast = n.s., luminal-HR - = 4.54 x 10 -302 , luminal-HR + = 0.00, lymph. EC = 2.83 x 10 -15 , lymphoid cells = 5.46 x 10 -12 , myeloid cells = 9.23 x 10 -8 ). G) Microscopic image of a duct stained against AR (red), AZGP1 (green), and ACTA2 (purple) in a trans-male (top) and cis-female (bottom) breast tissue of the CODEX microarray. H) Violin plot shows the GPAM co-expression module (GRNboost2, 95 th percentile, p-value, Wilcoxon < 2.22 x 10 -16 ) score in cis-female (purple) and trans-male (orange) adipocytes. I) Violin plot shows the TCF7L2 expression in trans-male (orange) and cis-female (purple) adipocytes (adjusted p-value, MAST = 3.48 x 10 -105 ) J) Volcano plot shows the differential expression of transcription factors in comparison of trans-male to cis-female adipocytes. Horizontal axis shows log 2 fold change in expression and the vertical axis shows –log 10 adjusted p-value. Purple data points indicate transcription factors with accessible chromatin matching the AR sequence motif (CisBP AR_689). K) Microscopic immunohistochemistry images show the staining against nuclei (DAPI; blue), adipocytes (PLIN1; green), and TCF7L2 (purple). Boxplot shows the median staining intensity of TCF7L2 among IHC scan-regions of cis-female (purple) and trans-male (orange) adipocytes (p-value, Wilcoxon = 0.0069).

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: RNA Expression, Expressing, Staining, Microarray, Sequencing, Immunohistochemistry

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) UMAP depicting fibroblast subclusters (top) in snRNA-seq data (matrix 1 and 2 = matrix-type fibroblasts, lipo-f = lipo-fibroblasts, vasc-f = vascular-like fibroblasts) and the distribution of patient samples in each gender identity (bottom). B) Heatmap shows scaled average expression of snRNA-seq markers identified for each of the fibroblast subclusters. C) Motif footprints for Androgen Receptor (AR, AR-CisBP M03389_2.00) among trans-male (orange) and cis-female (purple) fibroblast cells. Top panel shows the transposase bias-corrected signal, and the bottom panel shows the transposase bias. D) Right panel shows the enrichment of motifs among unique accessible chromatin peaks of trans-male (orange) and cis-female (purple) fibroblast cells. Left panel shows the fraction of the peaks of the corresponding cells which overlap with the motif. E) Boxplot shows the average ratio of androgen receptor (AR) staining intensity in fibroblast nucleus compared to the cytoplasm in tissue regions of cis-female (purple) and trans-male (orange) samples in CODEX microarray data (p-value, Wilcoxon = 1.2 x 10 -8 ). Dot colors indicate TMA origin of the shown regions F) Left panel boxplots show the fraction of cis-female (purple) and trans-male (orange) cells corresponding to 5 different classes of fibroblasts detected in tissue regions of CODEX microarray data (p-values, Wilcoxon: fibr-main = 0.00011, fibr-epi = 0.0046). Right panel shows the scaled staining intensities of various markers that distinguish the 5 subtypes of fibroblasts. G) Boxplots show per region average distance for each of the 5 subtypes of fibroblasts to the most proximal epithelial cell. H) Violin plots show the RNA expression of laminins LAMA2 (top) and LAMB1 (bottom) in fibroblast subclusters, split by cis-female (purple) and trans-male (orange) cells (adjusted p-values, MAST: LAMA2 in lipo-f = 2.60 x 10 -63 , matrix 1 = 5.11 x 10 -175 , matrix 2 = 1.03 x 10 -69 , vasc-f = 1.99 x 10 -10 ; LAMB1 in lipo-f = 3.71 x 10 -35 , matrix 1 = 1.46 x 10 -55 , matrix 2 = 3.08 x 10 -87 , vasc-f = 2.58 x 10 -13 ). I) AR binding sites (red markers) across genomic regions of LAMA2 and LAMB1 . Gene bodies are shown (light-green) with the promoter (arrow) and exon boundaries (dark-green). Genomic window shows chromatin accessibility in cis-female (purple) and trans-male (orange) fibroblasts. J) Boxplots show per-region average LAMA2 (left) and LAMB1 (right) staining intensities in the LAMB1 + fibroblast subtype on CODEX microarray data of cis-female (purple) and trans-male (orange) tissues. Dot colors indicate TMA origin of the shown regions. (p-value, Wilcoxon: LAMA2 = 0.64, LAMB1 = 0.0079) K) Microscopic images from CODEX microarray data show staining of LAMB1 (orange) among cis-female (left) and trans-male (right) tissues. Other markers include KRT8 (pan-luminal), TP63 (basal cells), and ACTA2 (basal cells). Arrow indicates distinctive LAMB1 ECM structures near epithelium and box highlights LAMB1 layer around acinus. L) Boxplots show the average RNA expression of ITGB1 among luminal-HR + , luminal-HR − , and basal epithelial cells of cis-female (purple) and trans-male (orange) samples (adjusted p-value, MAST in basal < 2.22 x 10 -16 ).

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: Expressing, Staining, Microarray, RNA Expression, Binding Assay

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) UMAPs show RNA-seq modules from (Lim et.al., 2009) overlaid on epithelial cells of snRNA-seq data. The corresponding flow cytometry markers used for detecting them are shown on the bottom. B) Boxplots show the fraction of each of the three epithelial cell types (basal, luminal, and luminal-HR + ) within each region of CODEX microarray data in cis-female (purple) and trans-male (orange) tissues. Dot colors indicate TMA origin of shown regions. C) The process of segmenting acini structures through masking luminal and basal cells (orange = KRT8 + KRT23, yellow = ACTA2 in left panel, respectively), segmenting the image, and expanding boundaries to estimate coverage of the smooth muscle layer. D) Boxplots show the average staining intensity of ACAT2, OXTR, and TP63 proteins among basal cells in tissue regions of cis-female (purple) and trans-male (orange) samples. (p-values, Wilcoxon: ACTA2 = 0.054, OXTR = 0.16, TP63 = 0.04) E) UMAP shows RNA-velocity streams over subclusters of basal cells (top) and the overlaid CellRank probability of terminal state (bottom). F) Scatterplot shows the log 2 fold change in expression of a given transcription factor (horizontal axis) and the difference in chromVAR z-score of the transcription factor (vertical axis) when comparison trans-male to cis-female basal cells. G) Volcano plot shows basal cell log 2 fold change in expression (horizontal axis) and −log 10 p-value (vertical axis) of genes annotated within the Reactome database pathway for cell junction organization. Purple data points contain a chromatin accessibility peak with a BACH2 transcription factor sequence motif (CisBP BACH2_113) match. Barplot shows the fraction of all genes (left) and Reactome pathway for cell junction organization genes (right) which contain a chromatin accessibility peak containing a BACH2 sequence motif match. H) UMAP plots of luminal-HR − cells in RNA space, showing detected subclusters (left) and distribution across cis-female and trans-male samples (right). I) Heatmap shows enrichment of luminal-HR − subclusters in marker pathways. Values show scaled average module scores. For each subcluster, the two most significant differentially expressed pathways were selected. (REAC = Reactome, WP = WikiPathways, BIOC = Biocarta, KEGG = Kyoto Encyclopedia of Genes and Genomes, PID = Pathway Interaction Database).

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: RNA Sequencing Assay, Flow Cytometry, Microarray, Staining, Expressing, Sequencing, Marker

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) Images from CODEX microarray data showing mammary acini structures from cis-female (top) and trans-male (bottom) tissues marked by expression of ACTA2 (basal cells; purple) TP63 (basal cell nuclei; blue) and KRT8 (luminal cells; green). B) Boxplots show per region average nucleus area among cis-female (purple) and trans-male (orange) epithelial cells (luminal-HR+, luminal cells, and basal) (p-values, Wilcoxon: luminal-HR+ = 0.00064, luminal = 0.016, basal = 0.54). Dot colors indicate TMA origin of shown regions. C) Boxplots show average area of acinar structures (left panel) and average area of acini border that was filled with ACTA2 signal (see ) among cis-female (purple) and trans-male (orange) tissues of CODEX microarray data. Dot colors indicate TMA origin of shown regions. (p-values, Wilcoxon: area = 0.026, ACTA2 coverage = 0.012) D) UMAP showing subclusters of basal cells in snRNA-seq data (top) and their distribution across trans-male and cis-female samples (bottom). E) RNA expression of ACTA2, OXTR (lactation markers) as well as TP63 in basal cells of trans-male (orange) and cis-female (purple) samples (adjusted p-values, MAST: ACTA2 = 8.86 x 10-296, OXTR = 9.59 x 10-262, TP63 = 1.16 x 10-96). F) Module scores of significantly enriched pathways overlaid over the UMAP of basal cells (REAC = Reactome, KEGG = Kyoto encyclopedia of genes and genomes). G) Right panel shows the enrichment of motifs among unique accessible chromatin peaks of trans-male (orange) and cis-female (purple) basal cells. Left panel shows the fraction of the peaks of the corresponding cells which overlap with the motif. H) Kernel density estimation and boxplot of module scores for selected significantly altered structural pathways in luminal-HR– cells (p-values, Wilcoxon: KEGG: adherens junction = 4.13 x 10-285, KEGG: focal adhesion = 1.42 x 10-255, KEGG: regulation of actin cytoskeleton < 1.42 x 10-255). I) Boxplots show average RNA expression (top) of integrin receptors from the “KEGG: regulation of actin cytoskeleton” pathway in luminal-HR– cells (adjusted p-values, MAST: ITGA2 = 4.89 x 10-201, ITGB8 = 6.40 x 10-267) and average expression of the ITGA2 and ITGB8 ligand FN1 in fibroblast subclusters and lymphatic endothelial cells (bottom) of trans-male and cis-female samples (adjusted p-values, MAST: matrix 1 = 1.66 x 10-54, matrix 2 = n.s., lipo-f = 1.32 x 10-16, vasc-f = n.s., lymph. EC = 3.13 x 10-99). J) Scatterplot shows Fisher’s exact test odds ratio (x-axis) and –log10 p-value (y-axis) corresponding to enrichment of each motif among the chromatin accessibility peaks for the genes of the “WikiPathways: focal adhesion pathway”. Colors indicate the log2 fold change in gene expression of the transcription factor corresponding to each motif. Gray motifs correspond to transcription factors without differential gene expression among luminal-HR– cells. Right barplot shows the fraction of all genes (left) and genes annotated within the focal adhesion pathway (right) which contain a chromatin accessibility peak matching the ESRRG sequence motif (cisBP ESRRG_697). K) Boxplot shows the ratio of stromal to epithelial cells in the epithelial neighborhood (see. ) among regions of cis-female (purple) and transgender male (orange) tissue in CODEX microarray data. (p-value, Wilcoxon = 0.0052). Dot colors indicate TMA origin of shown regions. L) The left microscopic image shows luminal (green KRT8-stained) and basal (purple ACTA2-stained) cells in a cis-female (left) and trans-male (right) breast tissue. The right schematic indicates the epithelial (blue), stromal (red), immune (green), and vasculature (orange) components of the left image.

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: Microarray, Expressing, RNA Expression, Sequencing, Staining

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) UMAP shows the sample identity of blood endothelial cells among the 18 samples without applied batch correction. Sample TM-9817 was found to be a strong outlier and was excluded from vasculature analysis. B) Gene modules from (Kalucka et.al., 2020) were used to define arteries (top), capillaries (middle), and veins (bottom) in snRNA-seq data. Scores are overlaid on the UMAP plot of blood endothelial cells. C) Heatmap shows column Z-score for average expression of the vasculature subcluster markers among the different subtypes of vasculature. D) UMAP shows staining subtypes of vasculature cells as determined from CODEX tissue microarray data. E) Heatmap shows row Z-score of vasculature marker staining score among the 5 staining subtypes of vasculature cells in CODEX tissue microarray data. F) Boxplot shows the fraction of different vasculature subtypes within the neighborhood of epithelial cells in cis-female (purple) and trans-male tissues. G) Boxplots show the fraction of lymphatic endothelial cells within snATAC-data of cis-female (purple) and trans-male (orange) samples (p-value, Wilcoxon = 0.021). H) Boxplots show the fraction of the different staining subtypes of vasculature in cis-female (purple) and trans-male (orange) tissue regions of CODEX microarray data. (p-values, Wilcoxon: lymph. EC = 0.019, endo-LNX1 + = 0.037, endo-main = n.s., endo-SMA = n.s., endo-immu. = n.s.) I) Barplot shows the most significantly enriched biological processes (BP) of the PPARG module in blood ECs. GO enrichment was done on the 95th percentile of PPARG co-expressed genes using g:Profiler. J) Kernel density estimate and boxplot of the Reactome VEGFR2-mediated cell proliferation pathway score in cis-female (purple) and trans-male (orange) lymphatic endothelial cells. (p-value, Wilcoxon = 1.06 x 10 -16 ) K) Selected significantly altered pathways among subclusters of blood endothelial cells are shown as pathway module scores of trans-male and cis-female cells.

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: Expressing, Staining, Microarray, Marker

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) UMAP showing subclusters of all myeloid (left) and lymphoid (right) cells detected in the snRNA-seq data. (CD8 = CD8 + T-Cells, CD4 = CD4 + T-Cells, T-effector = Effector T-Cells, NK = natural killer cells, mono.DC = monocyte derived dendritic cells, DC = dendritic cells) B) Boxplots show the fraction of main immune cell subtypes within entire immune compartment in each sample (GLM p-values, generalized linear model fitting a poisson: CD4 = 0.00035, CD8 = 4.035 x 10 -13 , T-effector = 0.045, NK = 0.00035, mono.DC = 0.017, macrophage = 0.52, monocyte = 0.055, DC = 0.0001). C) Boxplot shows the proportion of macrophages within the periphery of epithelial cells in cis-female (purple) and trans-male (orange) tissue regions of the CODEX microarray data. (p-value, Wilcoxon = 0.003) D) Kernel density estimates, and boxplots show the module scores of immune relevant Reactome pathways in macrophages of trans-male (orange) and cis-female (purple) samples. (p-values, Wilcoxon, class-I MHC mediated antigen processing/presentation = 8.32 x 10 -17 , clathrin mediated endocytosis = 3.64 x 10 -21 , roll like receptor TLR1 TLR2 cascade = 3.89 x 10 -16 ) E) Boxplots show the average RNA expression of PROS1 in basal cells of cis-female (purple) and trans-male (orange) samples (adjusted p-value, MAST = 3.17 x 10 -192 ). F) Volcano plot shows the average log 2 fold change and –log 10 adjusted p-value assessing the differential expression of genes in trans-male macrophages compared to cis-female macrophages. Purple data points indicate scavenger receptors. G) UMAP shows four immune cell staining sub classes (macrophage; red, immune endo.: green, immune-main: blue, and immune-epi.: orange) according to the staining pattern in CODEX microarray data. Size of the data points indicates the distance to the most proximal epithelial cell. Boxplot (below) summarizes the average distance of each group of immune cells to their most proximal epithelial cell. H) Microscopic images show staining of luminal (KRT8; green), basal (ACTA2; purple) and immune cells (CD45; red) within a trans-male (top) and a cis-female (bottom) breast tissue in CODEX microarray data. I) Microscopic image shows IHC staining of luminal (KRT8; red), immune (CD45; green), and T-lymphocyte (CD3; purple) cells within a trans-male (top) and a cis-female (bottom) breast tissue. White cells are double positive for CD45 and CD3. J) Boxplot shows the ratio of immune cells (CD45 + ) expressing CD3 to those not expressing CD3 (T-lymphocytes vs. other immune cells) within the epithelial neighborhood of cis-female (purple) and trans-male (orange) breast tissues of IHC scan regions.

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: Derivative Assay, Microarray, RNA Expression, Expressing, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) Heatmaps show scaled average RNA expression for markers of myeloid (left) and lymphoid (right) subclusters in the immune compartment. B) Boxplots show the proportion of the minor immune subclusters (B-cells and hematopoietic stem cells) among the immune cells of cis-female (purple) and trans-male (orange) samples. C) Boxplot shows the proportion of macrophages detected in CODEX microarray regions of cis-female (purple) and trans-male (orange) tissues. Only regions with at least 15 detected macrophages are included. Dot colors indicate which TMA the regions belong to. (p-value, Wilcoxon = 0.011) D) Heatmap showing scaled average staining intensity of immune cell markers among the 4 spatial subclasses of immune cells in CODEX microarray data. E) Boxplots show the proportion of immune cell spatial subclasses in cis-female (purple) and trans-male (orange) tissue regions of CODEX microarray data. (p-values, Wilcoxon: immune-main = 0.043, immune-epi. = 0.048, immune-endo = n.s.) F) Boxplot shows the ratio of immune cells (CD45 + ) expressing CD3 to those not expressing CD3 in IHC scan-regions in cis-female (purple) and trans-male (orange) tissues (p-value, Wilcoxon = 0.0014). G) Violin plots show the RNA expression of IL16 in matrix-2 and matrix-1 fibroblasts (top) and the RNA expression of TCF7 in CD4 + and CD8 + T lymphocytes among cis-female (purple) and trans-male (orange) cells. (adjusted p-values, MAST: IL16 in matrix-1 = 3.81 x 10 -10 , matrix-2 = 2.66 x 10 -01 , TCF7 in CD4 = 6.85 x 10 -08 , CD8 = n.s.) H) Barplot shows the most significantly enriched biological processes (BP) of genes upregulated in transgender male CD4+ T-cells.

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: RNA Expression, Microarray, Staining, Expressing

Journal: bioRxiv

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1101/2022.04.22.489095

Figure Lengend Snippet: A) Left microscopic image stained against nuclei (DAPI; blue) and adipocytes (PLIN1; green) and right shows the resulting nuclei segmentation. B) UMAP of adipocytes showing cis-female (top) and trans-male (bottom) cells colored by sample origin. C) Pathway enrichment (KEGG) of matched ligands and receptors that are differentially expressed between trans-male and cis-female samples in all mammary cell types. D) Boxplots show median INSR staining intensity in IHC scan-regions of cis-female (purple) and trans-male (orange) samples (p-value, Wilcoxon: 0.00043). Right shows IHC micrographs stained against nuclei (DAPI; blue), adipocytes (PLIN1; green), and INSR (red) in cis-female (left) and trans-male (right) tissues. E) Boxplots show the average staining intensity of INSR in cis-female (purple) and trans-male (orange) cell types in tissue regions of the CODEX microarray. Dot colors indicate the origin TMA of the shown regions (p-values, Wilcoxon: luminal = 0.0093, immune = 0.014). F) Boxplots show median NR4A1 staining intensity in IHC scan-regions of cis-female (purple) and trans-male (orange) samples (p-value, Wilcoxon: 7.5 x 10 -5 ). Right shows IHC micrographs stained against nuclei (DAPI; blue), adipocytes (PLIN1; orange), and NR4A1 (purple) in cis-female (left) and trans-male (right) tissues. G) Violin plots show the expression of PTEN (top), PIK3R1 (middle), and AKT3 (bottom) in cis-female (purple) and trans-male (orange) adipocytes (p-values, MAST: PTEN = 9.04 x 10 -96 , PIK3R1 = 5.75 x 10 -38 , AKT3 = 1.41 x 10 -18 ). H) Boxplots show the average staining intensity of AZGP1 in cis-female (purple) and trans-male (orange) epithelial cell types in tissue regions of the CODEX microarray. Dot colors indicate the origin TMA of the shown regions (p-values, Wilcoxon: luminal-HR+ = 0.0011, luminal = 0.00047, basal = 0.011). I) Pathway enrichment (WP) of GPAM co-expression module genes that are differentially expressed between trans-male and cis-female adipocytes. J) Volcano plot shows the average log 2 fold change and –log 10 adjusted p-value for differential expression of genes within the GPAM co-expression module among the trans-male and cis-female adipocytes. Purple data points indicate genes with a chromatin accessibility peak overlapping the TCF7L2 transcription factor sequence motif (CisBP TCF7L2_762) match. Barplots show the fraction of all genes (left) or genes within GPAM module (right) which contain a chromatin accessibility peak overlapping the TCF7L2 transcription factor sequence motif (purple).

Article Snippet: Conjugated antibodies were then titrated by performing CODEX staining on a TMA section using the full panel diluted at either 50x, 100x, or 200x.

Techniques: Staining, Microarray, Expressing, Sequencing

Journal: Communications Biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: a pDOC is a MITOMI-based microfluidic platform. The device includes flow (green) and control (Magenta) layers, and several modules: 1) common manifold enabling loading of materials, which in combination with microarray can enable up to 512 different experiments; 2) Parallel loading inputs enabling gel-like loading of up to 32 different experimental conditions; and 3) MITOMI module of 512 cell units, which controls the degradation assay process. b An illustration of two cell units. Each cell unit (marked by black dotted frame) comprises two chambers controlled by three pneumatic integrated valves. The total volume of each unit is about 1 nanoliter. The two chambers are separated by the ‘neck valve’ (I). Different cell units are separated via sandwich valves (II). Samples containing target proteins (IVT products) are loaded into the protein chamber and immobilized via biotinylated antibodies that can be either protein- or tag specific. The target proteins are trapped via MITOMI button valve (III) for quantification while the remaining unbound biomaterials are washed away. Flow direction within the device is indicated by green arrows. c Left, an image of a cell units within the MITOMI array. Target proteins can be immobilized to the protein chamber and quantified in several ways (illustrated on the right): i) An example of a target protein carrying a fluorescent tag (e.g., GFP) for detection (see green glowing tag) and a non-fluorescent tag for immobilization; ii) A target protein tagged with GFP for both immobilization (by anti-GFP antibodies) and detection; iii) The target protein is untagged. Detection is based on fluorescent lysine (Lys) incorporated during in vitro translation. Immobilization is via protein-specific antibodies. iv) The target protein is immobilized via tag- or protein-specific antibodies and detected by immunofluorescence via tag-specific antibodies coupled to fluorophore. Overall, on-chip immobilization of target proteins relies on biotinylated antibodies. Immobilization via non-biotinylated antibodies is possible if surface chemistry includes biotinylated IgG.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated anti-GFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Control, Microarray, Degradation Assay, In Vitro, Immunofluorescence

Journal: Communications Biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: a Time-dependent degradation of 35 S-labeled Flag-Securin-GFP, Flag-Geminin-GFP and their non-degradable variants (∆64 and ∆27, respectively) in NDB mitotic extracts (20 µl) supplemented with E-mix and Ubiquitin. Time-dependent proteolysis was resolved by SDS-PAGE and autoradiography. b Equivalent assays were performed with non-radioactive IVT products. Target proteins were incubated 1 h in reaction solution containing either extracts or PBS (control). Aliquots of each reaction mix (5 µl) were then loaded directly on a chip via separate channels, each containing dozens of cell units. Target proteins were immobilized to protein chambers via biotinylated anti-GFP antibody. The GFP tag was also used for quantification. GFP signals were calculated from 14–19 cell units per target protein per reaction condition (extracts vs. PBS). Box plots depict ratios of GFP signals (extracts/PBS) at t 60 min . Mean (x) and median (-) are indicated. * p value < 0.001. Representative raw data depicting detection on chip of the four target proteins are shown. c The degradation of Flag-Securin-GFP variant was assayed in tube as described in B. Here, however, aliquots were snap-frozen every 15 min. Time-lapse samples were then loaded on the chip for analysis. Target proteins were immobilized in protein chambers via anti-GFP antibodies and quantified based on GFP fluorescence. Time-dependent degradations of w.t vs. mutant Securin were quantified based on 35 S signal (standard analysis; n = 3] and GFP fluorescence (on-chip analysis). Plots depict mean signals and standard error bars. Mean signals were calculated from 26 cell units and normalized between max (1, t 0 ) and min ( 0 , t 60 min ) values, allowing proper comparison between two very different methods of detection. Error bars are shown. d Equivalent experiment to (c) performed with Flag-Geminin-GFP, except that immobilization was based on anti-Flag antibodies. n = 30–40 cell units.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated anti-GFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Labeling, Ubiquitin Proteomics, SDS Page, Autoradiography, Incubation, Control, Variant Assay, Fluorescence, Mutagenesis, Comparison

Journal: Communications Biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: a Degradation of Flag-Securin-Myc, Flag-Securin-GFP, and Green-Lys-labeled Flag-Securin (IVT products) in NDB mitotic extracts was assayed in tube for 1 h. On-chip analysis was performed in multiple ways: 1) Flag-Securin-Myc was immobilized by anti-Myc antibodies and detected by anti-Flag Cy5-conjugated antibodies; 2) Flag-Securin-GFP was immobilized and detected via the GFP tag; and 3) Green-Lys-labeled Flag-Securin was immobilized via anti-Flag antibodies and detected by the Green-Lys signal. Anti-Myc/Flag/GFP antibodies are biotinylated. Plots and raw data depict protein levels at t 0 vs. t 60 min . Signals are normalized to max values at t 0 . Mean values and standard error bars are shown; n = 20–40 cell units. * p value < 0.001. b Degradation of Green-Lys-labeled p27 (untagged) and Flag-Securin-GFP was assayed in S-phase extracts and analyzed by pDOC. p27 was immobilized via biotinylated anti-mouse IgG and anti-p27 antibodies. The Green-Lys signal was used for detection. Flag-Securin-GFP was immobilized via biotinylated anti-Flag antibodies. Following incubation with cell extracts, protein levels were quantified by GFP or Green-Lys fluorescence in 15 min intervals. Plots depict mean and standard error values normalized to max signal at t 0 . n = 20 (p27) and 10 (Securin) cell units.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated anti-GFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Labeling, Incubation, Fluorescence

Journal: Communications Biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: a Time-dependent degradation assay of 35 S-labeled Flag-Securin-GFP in NDB mitotic extracts. Assays were performed with undiluted IVT product (100%) or following 4x/10x dilution in reticulocyte lysate (25 and 10%, respectively). In all assays, 1 µl substrate was incubated in 20 µl cell extracts. Samples were snap-frozen in 20 min intervals and assayed by SDS-PAGE and autoradiography. b Equivalent degradation assays performed with non-radioactive Flag-Securin-GFP. Time-point samples were loaded on the chip for immobilization (via anti-GFP-biotinylated antibody) and detection (GFP fluorescence). The plots summarize data from three experiments, 15–20 cell units per experiment. Normalized mean and standard error values are shown (left). Representative raw data are shown on the right.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated anti-GFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Degradation Assay, Labeling, Incubation, SDS Page, Autoradiography, Fluorescence

Journal: Communications Biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: a , b Schematic illustration of complete on-chip degradation assay by pDOC. IVT protein products are immobilized to the surface of the ‘protein chamber’ via anti GFP biotinylated antibody (see more information in Fig. ). The closing of the MITOMI button valve traps the protein. All remains are washed away with PBS. Proper expression and immobilization of the target proteins are validated by scanning. Next, cell-free extracts are loaded into the extract chamber and trapped by closing the neck valve. The reaction begins with the opening of neck and MITOMI button valves, which allows diffusion of cell extracts into the ‘protein chamber’ and mixing with the target protein. The chip is placed on a 30 °C hot plate and scanned at 15 min intervals to provide kinetic information in real time. c GFP-tagged Securin and p27 IVT products were immobilized via anti-GFP-biotinylated antibody. Extract chambers were then filled with NDB mitotic extracts that support ubiquitination of Securin, but not of its non-degradable variant (Δ64) and p27 (negative control validating assay specificity). Protein degradation was assayed for 1 h during which the chip was scanned five times. The plot depicts mean GFP signals normalized to t 0 and standard error bars; n = 14–30 cell units. Representative raw data for p27 and Securin are shown on the right.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated anti-GFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Degradation Assay, Expressing, Diffusion-based Assay, Ubiquitin Proteomics, Variant Assay, Negative Control

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Cited2 is expressed broadly by CPN progenitors at E15.5, with expression refining to CPN of the somatosensory cortex by P3. A, Cited2 is highly expressed by CPN (red) relative to CSMN (blue) at critical times during development, as detected by microarray analysis of FACS-purified CPN and CSMN. Error bars denote SEM (Molyneaux et al., 2009). B, Western blot analysis showing that CITED2 protein is highly expressed as early as E15.5 in the neocortex, with expression decreasing postnatally, relative to a β-actin loading control. C–F, Expression of Cited2 is largely restricted to subpallial progenitors at E13.5 (C), but Cited2 is highly expressed in the cortical SVZ at E15.5 (D), the peak of superficial layer CPN birth, with expression maintained in layers II/III and V postnatally (E, F). G, Embryonically, Cited2 is expressed uniformly across the neocortex, detected across the SVZ at E18.5 (arrowheads) and across the cortical plate (CP). H, In the first days postnatally, however, its expression refines and becomes restricted to somatosensory cortex (arrows) by P3. I, At E15.5, Cited2 is highly expressed in the SVZ, extending into the intermediate zone (IZ). J, Cited2 (blue) is largely excluded from PAX6+ (green) radial glial progenitors of the VZ, but is highly expressed by TBR2+ (red) IPCs of the SVZ. K, Cited2 is largely excluded from the highly proliferative Ki67+ (green) VZ and apical mitotic cells, as indicated by pH3 (red), but is expressed by basally proliferating IPCs of the SVZ and IZ. Scale bars: C–F, 500 μm; G–I, 1 mm; C′–H′, J, K, 100 μm; I′, 200 μm. Dotted lines in J and K indicate apical, ventricular surface.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Expressing, Refining, Microarray, Purification, Western Blot

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Loss of Cited2 function results in specific reduction of TBR2+ IPCs in the E15.5 neocortex. A–A″, At E15.5, the peak of Cited2 expression, there is no change in the overall number of (PAX6+) radial glial progenitors in the Cited2;Emx1-Cre cKO neocortex compared with control littermates (WT). B–B″, There is, however, a significant reduction in the number of TBR2+ IPCs, which largely give rise to superficial layer CPN at this stage of development. C–D″, Reduction in TBR2+ IPCs might result from reduced proliferation of IPCs (C–C″), as indicated by proliferating cell nuclear antigen (PCNA, red) and TBR2 (green) double positivity (N = 11 WT, 6 cKO for A–C); specific reduction in basal cell divisions (D–D″), as indicated by position of pH3-positive mitotic cells (N = 10 WT, 5 cKO); and/or increased cell death (E), as indicated by expression of aC3. There is increased apoptotic cell death in the Cited2 cKO neocortex, both within the progenitor population and postmitotically (N = 11 WT, 6 cKO). F–F″, To directly investigate whether Cited2 cell-autonomously regulates proliferation of IPCs, we electroporated Cre recombinase and GFP into VZ progenitors of Cited2fl/fl and Cited2fl/wt littermates at E14.5 to excise Cited2 in a small subpopulation of neocortical progenitors. We used a BrdU pulse at E15.5, and immunocytochemistry for Ki67 at E16.5 to identify progenitors that continued to proliferate. There is a significant reduction in the number of Cited2-null (Cited2 fl/fl; Cre+) progenitors that incorporate BrdU at E15.5, or express Ki67+ at E16.5, demonstrating that Cited2-null IPCs are less likely to re-enter the cell cycle than their heterozygous counterparts. There is no change in the number of aC3+ cells in the Cited2-null progenitors at E16.5 (N = 4 Cited2fl/wt; 5 Cited2fl/fl). Scale bars, 100 μm. Error bars denote SEM. *p < 0.05; **p < 0.001 (Student's t test).

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Expressing, Immunocytochemistry

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Loss of Cited2 function results in reduced superficial layer thickness and total neocortical length at P3. A, At P6, the Cited2 cKO neocortex is smaller than in WT littermate controls, but both CPN (SATB2+) and CSMN (CTIP2+) are present and appropriately positioned. B, C, Anterograde labeling with DiI (B) and retrograde labeling with CTB (C) demonstrate that CPN are present and are targeting the contralateral hemisphere in the Cited2 cKO neocortex. However, both the distribution of retrogradely labeled CPN (C) and CUX1 (red, superficial layers) and CTIP2 (green, deep layers) immunocytochemistry (D) indicate that superficial layers are thinner in the Cited2 cKO neocortex, while the thickness of deep layers is unchanged (N = 4–5 per genotype for A–D). E, Quantitative analysis of neocortical layer thickness at P3 reveals that superficial layers (II–IV; LMO4, red) are significantly thinner in motor, somatosensory, and visual neocortical areas of P3 Cited2 cKO mice compared with those of control littermates. There is no change in deep-layer thickness (V, VI; CTIP2, green) in any region (N = 8 WT, 4 cKO). F, The Cited2 cKO cortex is visibly smaller than control littermates at P3, both in wholemount view and in sagittal sections. G, There is a significant reduction in total neocortical surface rostrocaudal length of ∼10%, as measured on four sagittal sections across the mediolateral axis (N = 8 WT, 4 cKO). Scale bars: A, 1 mm; B, 50 μm. Error bars denote SEM. *p < 0.05; **p < 0.001 (Student's t test).

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Labeling, Immunocytochemistry

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Neocortical surface length reduction is restricted to layers II/III of somatosensory cortex in the P3 Cited2 cKO neocortex. A–C, Analysis of three broad neocortical areas identified by LMO4 expression at P3 indicates a highly specific and substantial reduction (∼30%) in rostrocaudal surface length of the somatosensory area (blue) in the Cited2 cKO neocortex, entirely accounting for the total cortical surface length reduction (N = 8 WT, 4 cKO). D–G, Reduced somatosensory cortex length (black arrowheads) was confirmed via expression of multiple genes either excluded from superficial layers of the somatosensory cortex (D, E; Cadh8, EphA7), or specifically expressed in the somatosensory cortex (F; ephrinA5). Measurements of the acallosal layer IV somatosensory cortex (black arrows), by contrast (F, G; ephrinA5, Rorβ), reveals that there is no significant difference in non-CPN somatosensory cortex length in Cited2 cKO mice compared with that in WT mice (N = 8 WT, 4 cKO). H–H‴, In the P3 Cited2 WT neocortex, molecular markers of layer IV (green, RORβ; bracket) and the somatosensory cortex in layers II/III (red, Bhlhb5; white line) align at the motor/somatosensory border, shown in sagittal view (rostral to left). I–I‴, In the P3 Cited2 cKO neocortex, by contrast, the boundary of layer II/III expression of Bhlhb5 (white line, with additional low-level expression indicated by dashed line) is located caudal to layer IV RORβ expression (bracket), resulting in a misalignment of molecular areal boundaries between CPN of layer II/III and acallosal layer IV (H‴, I‴; N = 4–5 per genotype). Scale bars: D–G, 500 μm; H–I, 200 μm. Error bars denote SEM. *p < 0.05; **p < 0.001 (Student's t test).

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Additional loss of Lmo4 function does not alter Cited2 cKO neocortical thickness, but does reestablish layer II/III somatosensory neocortical length at the expense of the motor cortex. A–C, Additional loss of Lmo4 function in the Cited2-null neocortex (Cited2fl/fl;Lmo4fl/fl;Emx1Cre/+) does not alter the reduced reduction in superficial layer thickness of the Cited2-null motor or visual cortex, but does increase superficial layer thickness in the somatosensory cortex. Additional loss of Lmo4 function does not alter the overall reduction in total neocortical length (data not shown). D–G, Additional loss of Lmo4 function does, however, re-establish layer II/III somatosensory neocortical length (as measured by Bhlhb5 expression) to normal control length, at the expense of the layer II/III motor cortex. H, Length of motor (rostral to Bhlhb5 layer II/III expression), somatosensory (Bhlhb5 layer II/III positive), and visual (caudal to layer II/III Bhlhb5 expression) cortical areas was measured in control (Emx1-Cre negative), Lmo4 cKO (Cited2+/+;Lmo4fl/fl;Emx1Cre/+), Cited2 cKO (Cited2fl/fl;Lmo4+/+;Emx1Cre/+), and dcKO (Cited2fl/fl;Lmo4fl/fl;Emx1Cre/+) littermates. I–L, In the context of the shortened neocortical surface length in Cited2 cKO mice, additional loss of Lmo4 function re-establishes the length of the somatosensory area boundary, at the expense of the motor cortex length. By contrast, loss of Lmo4 function has no effect on the overall reduced neocortical length or layer II/III thickness of the Cited2 cKO neocortex. Scale bars: C, 100 μm; D–G, 1 mm. For each neocortical area, data were analyzed by a one-way ANOVA with Tukey's post-test. For all experiments, N = 14 controls, 7 Lmo4 cKO, 7 Cited2 cKO, and 8 double cKO mice. Error bars denote SEM. *p < 0.05, **p < 0.001, ***p < 0.0001.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Excision of Cited2 postmitotically via NEX-Cre does not alter neocortical laminar thickness or neocortical area lengths. A, B, NEX-Cre-mediated postmitotic excision of Cited2 does not visibly alter brain morphology or neocortical laminar development at P6 (A; as indicated by DAPI nuclear staining), and there is no significant difference in P3 neocortical length between Cited2; NEX-Cre cKO mice and control littermates (B). C–E, Further, there is no change in the overall neocortical laminar thickness of superficial or deep layers in the P3 Cited2; NEX-Cre cKO neocortex (C–C″), nor is there a change in the length of motor, somatosensory, or visual neocortical area lengths in the Cited2; NEX-Cre cKO neocortex (D, E). For all experiments, N = 4 WT, 4 cKO. Scale bars: A, 1 mm; C, 100 μm. Error bars denote SEM.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Loss of Cited2 function results in aberrant dendritic complexity of superficial layer somatosensory CPN. A, A′, Neuronal soma size (NeuN area) is not affected by loss of Cited2. Increased neuronal density is evident here, consistent with the modest, but significant, increase in cell density in layer II/III of the somatosensory cortex (23% increase, p = 0.01) quantified at P6 (see Results). N = 3 WT, 3 cKO. B–D, Dendritic complexity of layer II/III pyramidal neurons (primarily CPN) was analyzed at P22 by Golgi staining and Sholl analysis in the Cited2;Emx1-Cre cKO neocortex, compared with control littermates. B, B′, D, D′, There is no significant difference in dendritic complexity of layer II/III CPN in the motor cortex (B, B′) or visual cortex (D, D′) of Cited2; Emx1-Cre cKO mice, compared with littermate controls. C, C′, There is, however, a significant increase in dendritic complexity of layer II/III CPN in the somatosensory cortex of Cited2;Emx1-Cre cKO mice (two-way ANOVA p < 0.0001). E, CPN of the motor cortex are more complex than CPN of the somatosensory or visual cortex (two-way ANOVA p < 0.0001). F, In Cited2; Emx1-Cre cKO mice, dendritic complexity of somatosensory CPN is not significantly different from the CPN of the motor cortex (two-way ANOVA p = 0.16), suggesting that somatosensory CPN might be partially “motorized” in the absence of Cited2 function. G–I, Dendritic complexity of layer II/III CPN was analyzed at P22 by Golgi staining and Sholl analysis in a Cited2;NEX-Cre cKO neocortex, compared with control littermates. Following this postmitotic loss of Cited2 function, there is no change in dendritic complexity of superficial layer pyramidal neurons in any of the primary areas examined (motor, somatosensory, or visual cortices). Scale bars, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001, Bonferroni's post-test in B–F, G. B–E: motor, N = 17 WT, 12 cKO; somatosensory, N = 27 WT, 14 cKO; visual, N = 18 WT, 9 cKO. G–I: motor, N = 38 WT, 34 cKO; somatosensory, N = 37 WT, 20 cKO; visual, N = 34 WT, 19 cKO.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Interhemispheric CPN axonal connectivity is disrupted in the adult Cited2 cKO neocortex. A, B, HARDI tractography analysis of interhemispheric connections demonstrates a significant reduction in the size of the CC and in the number of callosal fibers in the juvenile/young adult (9 weeks old) Cited2; Emx1 cre cKO neocortex, compared with littermate controls, particularly within the mid-CC (corresponding to the somatosensory cortex). Reconstructed pathways are superimposed on the mean diffusion-weighted MRI of the brain. Pathways running between right and left are red; dorsal and ventral are green; and anterior and posterior are blue. The images do not include anterior commissure or olfactory bulb fibers; these were removed a priori to focus on the CC. Images in A′ and B′ similarly exclude hippocampal commissure fibers. N = 2 WT, 2 cKO. C, D, Staining for MBP in sagittal sections of Cited2 WT and cKO brains (C) followed by measurement of midsagittal CC area (D) identifies a reduction in CC area in Cited2 cKO brains, but demonstrates the structural integrity of the CC throughout all areas. N = 3 WT, 4 cKO. E–K, To investigate precision of callosal projections in the absence of Cited2 function, a focal injection of the anterograde tracer AAV-GFP was made in the somatosensory (extending into motor) neocortex at P6, and contralateral callosal projections were analyzed at 6 weeks of age. In contrast to the precise homotopic projections observed in Cited2 WTs (G), callosal projections in the Cited2 cKO somatosensory neocortex are diffuse (J). K, Relative GFP fluorescence intensity was measured in matched sagittal sections of the left injected neocortical hemisphere and in the same region in the contralateral projection hemisphere, demonstrating consistent caudal spread of callosal projections in the Cited2 cKO neocortex. N = 3 WT, 3 cKO. ANOVA analysis finds no change in the anterior tail (−1400 to −800 μm) or center (−700 to 700 μm) regions, but the posterior tail (800–1400 μm) of the cKO distribution is significantly different than that of the WT (p < 0.0001). Error bars denote SEM. *p < 0.05, (Student's t test) in C. *p < 0.001 (Bonferroni post-test) in K. Scale bars: A–B′, 1.5 mm; C, D, F, G, I, J, 1 mm; G′, J′, 500 μm.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Diffusion-based Assay, Staining, Injection, Fluorescence

Journal: Nature Communications

Article Title: Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane

doi: 10.1038/s41467-022-35708-1

Figure Lengend Snippet: a Representative TIRF images of CHO cells transfected with SNAP-GABA B1 (magenta), GABA B2 and eGFP-FLNA (green) for 24 h and labeled with SNAP-647. White color in the merge image is indicative of colocalization. b Quantification of GABA B and CD86 colocalization with FLNA. Shown are MCC values compared with those expected for homogenous distributions. Data are mean ± SEM. n = 11 and 20 cells (GABA B and CD86, respectively) examined over two independent experiments. c Scheme of the FLNA dimer with each subunit composed of an N-terminal actin-binding domain attached to 24 immunoglobulin-like domains. d Representative TIRF images of CHO cells co-transfected with SNAP-GABA B1 and Lifeact-GFP and either DsRed-FLNA17-18 or DsRed-FLNA19-20. e Quantification of the effect of FLNA19-20 on the colocalization between SNAP-GABA B1 and Lifeact-GFP. Shown are MCC values vs. those of homogenous GABA B1 distributions. Data are mean ± SEM. n = 29 and 32 cells (FLNA17-18 and FLNA19-20, respectively) examined over three independent experiments. f , g Representative confocal microscopy images of hippocampal neurons immunostained for GABA B1 (magenta) and either actin (green) ( f ) or FLNA (green) ( g ). Insets correspond to the regions delimited by the white boxes. Arrowheads, examples of colocalization. h , i Quantification of GABA B1 colocalization with actin ( h ) or FLNA ( i ) in hippocampal neurons. Shown are MCC values vs. those of homogenous GABA B1 distributions. Data are mean ± SEM. n = 7 and 8 cells (actin and FLNA, respectively) examined over three independent experiments. j Maximum-intensity projection of confocal microscopy stacks after 4X expansion microscopy of hippocampal neurons immunostained for GABA B1 (magenta) and FLNA (green). k Quantification of GABA B1 colocalization with FLNA based on 4X expansion microscopy images. Shown are MCC values vs. those of homogenous GABA B1 distributions. Data are mean ± SEM. n = 5 and 4 cells (GABA B1 and Bassoon, respectively) examined over two independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001 by two-tailed unpaired Welch’s t-test. ns statistically not significant. Scale bars, 5 µm (images in a , d ), 10 µm (images in f , g , j ), 2 µm (insets in f , g , j ). Source data are provided as a Source Data file.

Article Snippet: The Filamin A monoclonal antibody (M01), clone 4E10-1B2 was purchased from Abnova (H00002316-M01), the Bassoon mouse monoclonal antibody was from Enzo Life Sciences (NY, USA) (SAP7F407), the Bassoon rabbit polyclonal antibody was from Synaptic Systems (141003), goat Alexa Fluor-532 anti-mouse (A-11002) and goat Alexa Fluor-647 anti-rabbit (A-21245) were from Thermo Fisher Scientific, goat anti-rabbit IgG was from Sigma Aldrich (AP132), goat anti-rabbit CF568 was from Biotium (20801), the GAPDH mouse monoclonal antibody was from Invitrogen (AM4300), the DsRed mouse monoclonal antibody was from Santa Cruz (sc-390909) and the mouse IgG kappa binding protein coupled to HRP was from Santa Cruz (sc-516102).

Techniques: Transfection, Labeling, Binding Assay, Confocal Microscopy, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane

doi: 10.1038/s41467-022-35708-1

Figure Lengend Snippet: a Top, representative TIRF images of SNAP-GABA B1 (left) or SNAP-GABA B1 -IL1(mGluR2) (right) labeled with SNAP-647 in CHO cells additionally co-transfected with GABA B2 and Lifeact-GFP. Bottom, corresponding MCC analyses. Data are mean ± SEM. n = 35 and n = 8 (GABA B1 and GABA B1 -IL1(mGluR2), respectively) examined over three independent experiments. b Top, representative TIRF images of SNAP-GABA B1 (left) or SNAP-GABA B1 -IL1(mGluR2) (right) labeled with SNAP-647 in CHO cells co-transfected with GABA B2 and eGFP-FLNA. Bottom, corresponding MCC analyses. Data are mean ± SEM. n = 11 and n = 7 (GABA B1 and GABA B1 -IL1(mGluR2), respectively) examined over two independent experiments. c , Peptide microarray results. Peptide microarrays encompassing GABA B1 and GABA B2 intracellular domains were incubated with increasing concentrations of cell lysates containing DsRed-FLNA19-20. The fluorescence readout is plotted as a heatmap, normalized to the strongest detected signal. A linear dependency between signal intensity and lysate concentration was observed. d Screening with a microarray containing single-point alanine mutations of the GABA B1 IL1. Data are presented as in c . e Cartoon representation of GABA B inactive structure (PDB ID: 6WIV) highlighting the IL1 as sticks and the amino acids involved in the FLNA interaction in red. Experiments in c and d were performed twice for each condition, each time in duplicate. *** p < 0.001, **** p < 0.0001 by two-tailed unpaired Welch’s t -test. Scale bars, 10 µm. Source data are provided as a Source Data file.

Article Snippet: The Filamin A monoclonal antibody (M01), clone 4E10-1B2 was purchased from Abnova (H00002316-M01), the Bassoon mouse monoclonal antibody was from Enzo Life Sciences (NY, USA) (SAP7F407), the Bassoon rabbit polyclonal antibody was from Synaptic Systems (141003), goat Alexa Fluor-532 anti-mouse (A-11002) and goat Alexa Fluor-647 anti-rabbit (A-21245) were from Thermo Fisher Scientific, goat anti-rabbit IgG was from Sigma Aldrich (AP132), goat anti-rabbit CF568 was from Biotium (20801), the GAPDH mouse monoclonal antibody was from Invitrogen (AM4300), the DsRed mouse monoclonal antibody was from Santa Cruz (sc-390909) and the mouse IgG kappa binding protein coupled to HRP was from Santa Cruz (sc-516102).

Techniques: Labeling, Transfection, Peptide Microarray, Incubation, Fluorescence, Concentration Assay, Microarray, Two Tailed Test

Journal: Nature Communications

Article Title: Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane

doi: 10.1038/s41467-022-35708-1

Figure Lengend Snippet: a Representative frame of a single-molecule TIRF image sequence of SNAP-GABA B1 labeled with SNAP-647 in CHO cells co-transfected with GABA B2 and DsRed-FLNA19-20 or DsRed-FLNA17-18 as negative control. Images are representative of at least three independent experiments. b Representative outcome of single-particle tracking from at least three independent experiments. Each detected particle is surrounded by a blue circle and particle trajectories are shown in magenta. c Diffusion coefficients of GABA B receptor particles in the four groups identified by the TAMSD analysis. Data are mean ± SEM from n = 16 and 15 cells (2809 and 2670 trajectories) for FLNA17-18 and FLNA19-20, respectively, examined over three independent experiments. d Single-molecule analysis of GABA B -FLNA interactions. CHO cells were co-transfected with SNAP-GABA B1 and CLIP-FLNA and labeled with SNAP-647 and CLIP-TMR, respectively. A representative example of a transient colocalization event between a GABA B and a FLNA molecule is shown. e – g Relative frequency ( e ), duration ( f ), and density of diffusivity states ( g ) of single-molecule colocalizations between FLNA and either GABA B1 or GABA B1 -IL1(mGluR2) under basal and stimulated conditions (GABA 100 µM; 5 min incubation). Data are mean ± SEM. n = 53 (GABA B1 basal), 20 (GABA B1 stimulated), 23 (GABA B1 -IL1(mGluR2) basal) and 12 (GABA B1 -IL1(mGluR2) stimulated) examined over three independent experiments. * p < 0.05, ** p < 0.01 by two-tailed Mann-Whitney U test. ns statistically not significant. Scale bars, 5 µm ( a ), 500 nm ( b , d ). Source data are provided as a Source Data file.

Article Snippet: The Filamin A monoclonal antibody (M01), clone 4E10-1B2 was purchased from Abnova (H00002316-M01), the Bassoon mouse monoclonal antibody was from Enzo Life Sciences (NY, USA) (SAP7F407), the Bassoon rabbit polyclonal antibody was from Synaptic Systems (141003), goat Alexa Fluor-532 anti-mouse (A-11002) and goat Alexa Fluor-647 anti-rabbit (A-21245) were from Thermo Fisher Scientific, goat anti-rabbit IgG was from Sigma Aldrich (AP132), goat anti-rabbit CF568 was from Biotium (20801), the GAPDH mouse monoclonal antibody was from Invitrogen (AM4300), the DsRed mouse monoclonal antibody was from Santa Cruz (sc-390909) and the mouse IgG kappa binding protein coupled to HRP was from Santa Cruz (sc-516102).

Techniques: Sequencing, Labeling, Transfection, Negative Control, Single-particle Tracking, Diffusion-based Assay, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . UMAP embedding of Fibroblast/Pericyte/Myofibroblast cells from 13 human kidneys (n=2,689). Colors represent the cell types. Lines refer to a lineage trajectory predicted by slingshot (see ). b . Expression of selected genes on the embedding of a. c . Gene Ontology Biological Process analysis for Pericyte (Pe), Myofibroblast (MF), Fibroblast cell clusters (Fib) and vascular smooth muscle cells (VSMCs) based on the top marker genes for each cluster (CD10 - data, see ). d . ECM score and scaled expression of select genes visualized on the Mesenchymal cell Diffusion Map embedding of . e.-h . The distribution of ECM score, collagen score, glycoprotein score and proteoglycan scores stratified by epithelial cell clusters in the CD10 - - cell fraction. i . Scaled expression of select genes in proximal tubules and injured proximal tubule cell clusters. Each 100 cells are averaged in one column. j . Gene Ontology Biological Process analysis based on differential expression between proximal tubules and injured proximal tubules. k.-n . The distribution of ECM score, collagen score, glycoprotein score and proteoglycan scores for epithelial cells (CD10 + cell fraction) o . Percentage of cells expressing PDGFRb and Col1a1 in each main cell niche. Neuronal Schwann cells were excluded since they are represented by a small number of cells.

Article Snippet: Cells were labeled with the following antibodies: anti-CD10 human (clone HI10a, biolegend, 1:100), anti-PDGFRb mouse (clone PR7212, R&D, 1:100), anti-PDGFRalpha mouse (clone APA5, biolegend, 1:100)), anti-CD45 mouse (clone 30_F11).

Techniques: Expressing, Marker, Diffusion-based Assay

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . Patient samples (n=8) visualized on the UMAP from . Different cell clusters are indicated by different colors. Stratification of single cells according to patient clinical parameters (CKD=chronic kidney disease, eGFR=estimated glomerular filtration rate). b-c . Expression of select genes on the same UMAP embedding from a. d . Scaled gene expression of the top 10 genes in each cell type/state cluster. Gene ranking per cell cluster was determined by genesorteR. e . Correlation between cell clusters identified in CD10 - data ( , columns) and PDGFRb + data ( , rows). f . ECM score stratified by 4 main cell types in PDGFRb + data. g . ECM score stratified by main mesenchymal cell types. h . ECM score stratified by 5 epithelial cells clusters. i . ECM score visualized on the UMAP embedding from a. j . Doublet Score (see ) for human PDGFRb + cells. k . Representative image of combined immunofluorescent and multiplex RNA in-situ hybridization of LTA (proximal tubular marker), Col1a1 and PDGFRb + . Note Col1a1 and PDGFRb expression in LTA + tubular cells (j’ arrows). l . Representative image of combined immunofluorescent and multiplex RNA in-situ hybridization of CD68 (macrophage marker), Col1a1 and PDGFRb. m . Representative image of multiplex RNA in-situ hybridization of Pecam1, Col1a1 and PDGFRb. Scale bars k-m 50 μm.

Article Snippet: Cells were labeled with the following antibodies: anti-CD10 human (clone HI10a, biolegend, 1:100), anti-PDGFRb mouse (clone PR7212, R&D, 1:100), anti-PDGFRalpha mouse (clone APA5, biolegend, 1:100)), anti-CD45 mouse (clone 30_F11).

Techniques: Filtration, Expressing, Multiplex Assay, RNA In Situ Hybridization, Marker

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . KEGG pathway enrichment analysis along pseudotime for lineage 1 (see .) b . Top: Gene expression dynamics along pseudotime for lineage 2 (Fibroblasts to Myofibroblasts, see .). Cells (in columns) were ordered along pseudotime and genes (in rows) that correlate with pseudotime were selected and plotted along pseudotime (see ). Each 10 cells were averaged in one column. Genes were grouped signifying their pseudotime expression pattern. Selected example genes for each group are indicated. See Supplementary File 3 for gene cluster assignments. Bottom: Cell cycle stage along pseudotime as percent of each 500 cells along pseudotime. c . Same as in b. but for lineage 3 cells (see ) d . PID signaling pathway enrichment analysis along pseudotime for lineage 2 cells ordered along pseudotime as in b. e . KEGG pathway enrichment analysis along pseudotime for lineage 2. f . Same as in d. but for lineage 3 cells (see ). g . Same as in e. but for lineage 3 cells. h.-k . Violin plots across mesenchymal cells types of selected genes of the human PDGFRb + dataset in . l . TF scores for proximal promoter regions (l) and distal regions (m) obtained by TF sequence motif enrichment analysis for top marker genes for the mesenchymal cell clusters of the human PDGFRb + dataset (see ). Note enrichment of Fos and Jun motifs in promoters of fibroblast marker genes. m . Schematic of human kidney PDGFRb + cell generation and immortalization. n . Cell proliferation (WST-1) and expression of cFos, Col1a1, Postn and Ogn by RNA qPCR after AP-1 inhibitor treatment (T-5224) and/or TGFb treatment of immortalized human PDGFRb kidney cells. n=3 per group. *P < 0.05, **P<0.01, *** P < 0.001, ****P <0.0001 by 1-way ANOVA followed by Bonferroni’ post-hoc test. Mean± S.D. o . Expression of Ogn (Fib1+3) and Postn (MF1) visualized on the same UMAP embedding from . p . AP-1 average TF expression (left) and average expression of putative AP-1-regulated genes (right) against Collagen scores stratified by fibroblast and myofibroblast cells. Interestingly, the expression of AP-1 anti-correlates with collagen score but the expression of its target genes positively correlates with collagen score, potentially pointing towards an inhibitory role for AP-1. q . The number of statistically significant receptor-ligand interactions between mesenchymal cells and all other cell types (CD10- fraction, ) according to CellphoneDB Analysis. Dendritic cells, monocytes, myofibroblasts, podocytes, arteriolar endothelial cells and injured tubules as major sources of signaling ligands to pericytes fibroblasts and myofibroblasts. r . Dot plot for significant ligand-receptor interactions from the selected signaling pathways EGFR, PDGF, WNT, TGFb, Notch and Hedgehog for pericytes, fibroblast and myofibroblasts. Interacting ligand-receptor and cell types are shown by pairs. The first cell type of the interacting pair expresses the ligand and the second cell type expresses the receptor (i.e. first and second proteins from the interaction, respectively). Ligand-receptor interactions are grouped by signaling pathways. Yellow: EGFR, pink: PDGF, green: WNT, red: TGFb, black: Notch, blue (light or dark): mixed of TGFb and EGFR. None of the hedgehog interactions were significant. For details on statistics and reproducibility, please see .

Article Snippet: Cells were labeled with the following antibodies: anti-CD10 human (clone HI10a, biolegend, 1:100), anti-PDGFRb mouse (clone PR7212, R&D, 1:100), anti-PDGFRalpha mouse (clone APA5, biolegend, 1:100)), anti-CD45 mouse (clone 30_F11).

Techniques: Expressing, Sequencing, Marker

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . Representative image of Col1a1 in-situ hybridization in a PdgfrbCreER;tdTomato kidney after UUO surgery. Scale bar 10 μm b-c . Quantification of aSMA + cells in PDGFRbtdtom + kidneys from UUO day 10. n=3. Mean± SD. d . Scaled expression of the top 10 genes by specificity in each cell cluster depicted in . All cells from each cell cluster are averaged in one column. e . Expression of select genes in all 10 cell clusters from . f . ECM score visualized on the same UMAP embedding from . g . Distribution of ECM score, collagen score, glycoprotein score and proteoglycan score per cell cluster. h . Immunofluorescence (IF) staining in sham and UUO (day 10) mouse kidney showing Pdgfra expression in a subset of PDGFRbCreER;tdTomato positive cells (arrows). i . RNA in-situ hybridization showing colocalization of Col1a1 expression in PDGFRa/PDGFRb double-positive cells. Col1a1/PDFGRa/PDFRb triple-positive cells (arrows) occur solely in the kidney interstitium. j . Left: Col1a1 expression and ECM score in CD10 negative cells stratified according to PDGFRa and PDGFRb expression. Right: Percent of Col1a1 positive and negative cells in the same data, stratified in the same way. Col1a1 negative cells occur mostly in PDGFRa/b double-negative cells while Col1a1 positive cells occur predominantly in PDGFRa/b double-positive cells (n=51,849). Group comparisons: (other genes) vs. (a/b): p~0, (a - ) vs. (a/b): p~0, (b) vs. (a/b): p~0, (other genes) vs. (a): p~0, (b) vs. (a): p~0, (other genes) vs. (b):p~0. Bonferroni corrected p-values based on a two-sided Wilcoxon rank sum test. k . Distribution of IF/TA-Score over 62 patients and representative image of a trichrome stained human kidney tissue microarray (TMA) stained by multiplex RNA in-situ hybridization using PDGFRa, PDGFRb and Col1a1 probes with nuclear counterstain (DAPI) of 62 kidneys (patient data in Extended Data Table 2) (left), average scaled Col1a1 expression in the in-situ hybridization data stratified by PDGFRa/PDGFRb detection in the same data (middle) and percent of Col1a1 positive and negative cells in the same data stratified in the same way (right). Group comparison: (a/b) vs. (col1α1): p~0, (a/b) vs. (b): p~0, (a/ - ) vs. (a): p~0. Bonferroni corrected p-values based on a two-sided Wilcoxon rank sum test. l-p . A Diffusion Map embedding of pericytes and matrix producing cells with annotation of the different time points in m, cell cluster annotation in n and scaled expression of selected genes in o-q. q. The surgery type per cell (sham versus UUO) visualized on the same UMAP embedding from (top), or with colors representing the cell types/states (bottom). r . Expression of select genes on the same UMAP embedding from 3j. s . ECM and collagens score distribution for the 4 major cell types (top) and for mesenchymal clusters (bottom). Scale bars h+j 50 μm, in k 10 μm. For details on statistics and reproducibility, please see .

Article Snippet: Cells were labeled with the following antibodies: anti-CD10 human (clone HI10a, biolegend, 1:100), anti-PDGFRb mouse (clone PR7212, R&D, 1:100), anti-PDGFRalpha mouse (clone APA5, biolegend, 1:100)), anti-CD45 mouse (clone 30_F11).

Techniques: In Situ Hybridization, Expressing, Immunofluorescence, Staining, RNA In Situ Hybridization, Microarray, Multiplex Assay, Diffusion-based Assay

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . Classification tree of human PDGFRb dataset derived by the CHETAH algorithm based on single cell expression and clustering information. b . Supervised classification of mouse PDGFRa + /b + cells using human PDGFRb + cells as a reference (see classification tree in a.). Heatmap displays percentage of mouse PDGFRa + /b + cells in each mouse cell cluster. Fibroblasts 1 in mice are largely classified as Fibroblasts 1 according to human data. Mouse myofibroblasts are classified as Node 15 and myofibroblasts 2b in humans indicating variability between mouse and human with myofibroblast states. c . Schematic of proposed cellular origin of fibrosis. d . Scaled gene expression of transcription factors discovered by ATAC-Seq (see ) in six fibroblasts and myofibroblast cell populations. e . ATAC-Seq signal for motif matches inside open chromatin regions for five selected transcription factors. f . Genome browser snapshots for select genes. ATAC-Seq signal and motif matches in open chromatin regions are shown. Multiz Align is conservation scores between mouse and human, ClinVar lift is clinical variants lift to mouse genome coordinates. Nrf, Irf8, Elf/Ets and Klf motifs are located in promoter and enhancer open chromatin regions of myofibroblast associated genes such as Col1a1, Col15a1, Tgfb and Nkd2. Creb5_Atf3 is found in genes associated to Fib1. cluster, such as Tmeff2. g . Expression of some of the genes investigated in g-i. Visualized on the same UMAP embedding from . i . Scaled expression of genes that correlate or anti-correlate with injury time across matrix producing cells (mouse PDGFRb + data). Note the expression of Ogn, Scara5 and Pcolce2 is largely specific to day0-day2 cells while the expression Nkd2, Fbn2 and Nkd1 is specific is increased in day 10 after UUO. h . Signaling pathway enrichment in the same mesenchymal cell clusters depicted in .

Article Snippet: Cells were labeled with the following antibodies: anti-CD10 human (clone HI10a, biolegend, 1:100), anti-PDGFRb mouse (clone PR7212, R&D, 1:100), anti-PDGFRalpha mouse (clone APA5, biolegend, 1:100)), anti-CD45 mouse (clone 30_F11).

Techniques: Derivative Assay, Expressing

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . Expression of Nkd2 visualized on the UMAP embedding from . b . Percent of Col1a1+/- cells in mouse Pdgfra+/Pdgfrb+ cells ( , stratified by Pdgfra and Nkd2 expression). c . Scaled gene expression of Nkd2 correlating or anti-correlating genes in human Pdgfrb+ cells . d.-e . RNA in-situ hybridization (ISH) of PDGFRa, PDGFRb and NKD2 in human kidneys and quantification of triple positive cells (n=36, Patient data in Extended Data Table 2). n=20 and 16. Two-tailed Mann-Whitney test. Tukey box whisker plot. IF-score = interstitial fibrosis score. Scale bar 10μm. f.-g . Representative Western blots of Nkd2 overexpression and KO cells. For gel source data, see . h . GSEA (Gene set enrichment analysis) of ECM genes in Nkd2-perturbed PDGFRb - kidney cells. n=3 each. * P < 0.05, **p< 0.01, and ***p < 0.001 as determined by FGSEA-multilevel method after adjusting p-values for multiple testing correction (Benjamini & Hochberg). i . ISH of Pdgfra, Pdgfrb and Nkd2 in human iPSC derived kidney organoids. j . Quantification of Nkd2 RNA expression in organoids. n=4 each. Two-tailed unpaired t-test. k.-l . Immunofluorescence staining and quantification of Col1a1 in organoids. n=4 each. * P < 0.05, **p< 0.01, and ***p < 0.001 by 1-way ANOVA followed by Bonferroni’s correction. Scale bar in i+k 50 μm. Data shown as mean±SD. For details on statistics and reproducibility, please see .

Article Snippet: Cells were labeled with the following antibodies: anti-CD10 human (clone HI10a, biolegend, 1:100), anti-PDGFRb mouse (clone PR7212, R&D, 1:100), anti-PDGFRalpha mouse (clone APA5, biolegend, 1:100)), anti-CD45 mouse (clone 30_F11).

Techniques: Expressing, RNA In Situ Hybridization, Two Tailed Test, MANN-WHITNEY, Whisker Assay, Western Blot, Over Expression, Derivative Assay, RNA Expression, Immunofluorescence, Staining

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a-b . Gene ontology Biological Process terms for genes that correlate or anti-correlate with Nkd2 + expression across single cells in pericytes fibroblasts and myofibroblasts in mouse PDGFRa + /b + data (a) and human PDGFRb + data (b). Genes correlated with Nkd2 + expression are related to ECM expression, integrin signaling and focal adhesion. c . Pathway activity as estimated by the PROGENy algorithm in NKD2 + vs. NKD2 - cells from the human PDGFRb + dataset. p>0.05 n.s., *p<0.05, **p<0.01, ***p<0.001, p values were adjusted for multiple testing using Benjamin/Hochberg method (FDR) (c). d . Scaled gene expression of top 100 genes whose expression is correlated or anti-correlated with Nkd2 expression across single cells in human PDGFRb + data (see also b.) e. Gene regulatory network predicted based on the expression of cells and genes depicted in l. using the GRNBoost2 + algorithm. Connection between genes indicate putative direct or indirect regulatory interactions. Colors indicate clustering of the gene regulatory network using the Louvain algorithm and highlights the regulatory network of ECM expression (module 2, Nkd2 + ) and fibroblast and pericyte maintenance (module 4 and 3) f . Module 2 from l. Depicted separately, connections of Nkd2 are highlighted in red. g . Expression of genes highlighted in e. and f. including Etv1 transcription factor and Lamp5 which are both directly connected to Nkd2 in e. and f. h . Expression of Col1a1, Fibronectin (Fn) and Acta2 (aSMA) by qPCR after Nkd2 over-expression in human immortalized PDGFRb + cells treated with transforming growth factor beta (TGFb) or vehicle (PBS). n=3 per group. 1-way ANOVA followed by Bonferroni’ post-hoc test. Data represent the mean ± SD. i . Expression of NKD2 by RNA qPCR in NKD2 KO cells. ****P <0.0001 by 1-way ANOVA followed by Bonferroni’ post-hoc test. Data represent the mean ± SD. j . Expression of Col1a1, Fibronectin (Fn) and Acta2 by RNA qPCR after Nkd2 knock-out in the same clones depicted in h. n=3 per group. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 (vs. control NTG); ****p <0.0001 (vs. TGFb NTG) by 2-way ANOVA followed by Sidak’s post-hoc test. Data represent mean ± SD. k . PID signaling pathways enriched in PDGFRb + NKD2-KO clones and overexpression (up indicates up-regulated genes in indicated condition, and down indicates down regulated genes). l . Gene ontology Biological Process terms enriched in PDGFRb + NKD2-KO clones (up indicates up-regulated genes in KO condition, and down indicates down regulated genes). m . Scaled gene expression of WNT pathway receptors and ligands in Nkd2-perturbed human kidney PDGFRb + cells.*p< 0.05, **p< 0.01, and ***p < 0.001 as determined by the empirical Bayes from the test for differential expression after adjusting p-values for multiple testing correction (Benjamini & Hochberg) n . Representative image of multiplex RNA in-situ hybridization of PDGFRa, PDGFRb and NKD2 in human iPSC derived kidney organoids. o . Immunofluorescence stainings of human iPSC derived kidney organoids (day 7+18). LTA and HNF4a mark proximal tubular like-cells. pan-CK (Cytokeratin) marks epithelial-like cells. ERG (ETS regulated-gene) marks endothelial-like cells. Dach1 and Nephs1 mark podocyte-like cells. Col1a1 marks fibroblast/myofibroblasts. p . Immunofluorescence stainings of Col1a1 in IL1b treated kidney organoids. Scale bar in n, o, p=50 μm. For details on statistics and reproducibility, please see .

Article Snippet: Cells were labeled with the following antibodies: anti-CD10 human (clone HI10a, biolegend, 1:100), anti-PDGFRb mouse (clone PR7212, R&D, 1:100), anti-PDGFRalpha mouse (clone APA5, biolegend, 1:100)), anti-CD45 mouse (clone 30_F11).

Techniques: Expressing, Activity Assay, Over Expression, Knock-Out, Clone Assay, Multiplex Assay, RNA In Situ Hybridization, Derivative Assay, Immunofluorescence

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: Genome-wide Binding by MITF (A) Genome browser screenshots derived from ChIP-seq using anti-HA antibody of 501mel cells stably expressing ectopic HA-tagged MITF. (B) Consensus motif for the most significant 900 genome-wide MITF-binding sites predicted from 60-bp regions around peak summits generated by MEME. (C) The proportion of peaks with or without a 5′-TCA(T/C)GTGN-3′ motif at different peak heights. (D) Relationship between motif frequency and peak height as in (C). (E) Sequences associated with a selection of differentiation or non-differentiation-associated MITF target genes. (F) Box and whisker plots of peak height related to motif. Center of notches indicates the median. Green box indicates range of peak heights within which lie a set of well-characterized differentiation-associated genes in addition to many other non-differentiation genes. See also Figure S1 and .

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Genome Wide, Binding Assay, Derivative Assay, ChIP-sequencing, Stable Transfection, Expressing, Generated, Selection, Whisker Assay

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: MITF Can Be Acetylated (A) Indicated expression vectors were transfected into Phoenix cells and input and anti-FLAG immunoprecipitates western blotted. (B) Western blot of 501mel cells treated with 200 nM TPA for indicated times. (C) Western blot of extracts from cells transfected with BRAF and/or p300 expression vectors. (D) Western blot of Phoenix cells transfected with indicated vectors and HA-MITF, ±20 μM U0126 immunoprecipitated using anti-HA antibody. (E) Schematic showing the melanocyte-specific MITF-M(+) isoform. The five acetylated lysine residues identified in MITF-M peptides by mass spectrometry are indicated below. ERK, p38, and RSK phosphorylation sites are indicated above with the CBP/p300-binding site. (F) MITF DNA-binding domain-DNA co-crystal structure showing the MITF K243-DNA phosphate-backbone contact. (G) Conservation of K243 between bHLH and bHLH-LZ family members. (H) Peptide array containing indicated residues as 14-amino-acid peptides immobilized on a cellulose membrane probed with rabbit anti-acetyl-K243 antibody. (I) Western blot using anti-acetyl K243 or anti-MITF antibodies of immunoprecipitated GFP-MITF expressed alone or with co-transfected CBP or p300. (J) Western blot using anti-acetyl K243 or anti-MITF antibodies of HIS-tagged MITF purified with nickel beads. All samples were from the same blot. See also Figure S2 .

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Mass Spectrometry, Phospho-proteomics, Binding Assay, Peptide Microarray, Membrane, Purification

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: K243 Status Determines MITF DNA-Binding Affinity (A) Comparison of circular dichroism (CD) spectra of bacterially expressed and purified MITF WT and mutant DNA-binding domains. The mean residue ellipticity is plotted in dg × cm 2 × dmol −1 against the wavelength (in nm). CD spectra show the mutations cause no major structural changes. (B) DNA-binding affinity of bacterially expressed and purified MITF WT and mutant DNA-binding domains determined using fluorescence anisotropy. Representative titration curves of each fluorescein-labeled oligonucleotide with MITF WT and mutants. The anisotropy values are the average of triplicate measurements from which the baseline corresponding to the anisotropy of the free fluorescent probe was subtracted. (C) The dissociation constants of MITF WT and mutants on oligonucleotides containing four different recognition sequences determined by fluorescence anisotropy.

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Binding Assay, Comparison, Circular Dichroism, Purification, Mutagenesis, Residue, Fluorescence, Titration, Labeling

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: K243 Controls MITF Function In Vivo (A) Complementation of neural crest MITFa- null nacre zebrafish using MITF WT and K238 (equivalent to K243 in human MITF) mutants (left) and quantification of numbers of melanocytes (right). The dots in the plots represent numbers of melanocytes in each rescued embryo with at least one melanocyte. See also Figure S3 . (B) Western blot of 501mel cells stably expressing HA-MITF WT and mutants (from the same gel). (C) Tumor formation after subcutaneous inoculation of indicated cell lines into athymic nude mice. (D) Example tumors. (E) Tumor size over time using indicated cell lines. Error bars indicate S.E.M.

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: In Vivo, Western Blot, Stable Transfection, Expressing

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: K243 Status Determines MITF Genome-wide Distribution (A) Heatmap of MITF WT and K243 mutant average tag density derived from two biological replicate ChIP-seq experiments of HA-tagged MITF expressed using 0 or 20 ng doxycycline centered on WT occupied regions (20 ng doxycycline). (B) Numbers of ChIP peaks called using HA-tagged MITF WT or mutants induced using 0 or 20 ng doxycycline. See also . (C) Read coverage of two replicates for each of the WT and K243 mutant ChIP-seq experiments expressed using 0 or 20 ng doxycycline centered around peak coordinates of the WT at 5-bp binning intervals. Numbers on the x axis indicate distance from center of the peak (in bp). (D) Genome browser screenshots of indicated loci showing HA-tagged WT and mutant MITF ChIP-seq profiles from iMITF cell lines expressing HA-tagged MITF at 0 or 20 ng doxycycline as indicated. (E) Box and whisker plots showing peak score for two replicate (R1 and R2) ChIP-seq experiments for the WT and two K243 mutants related to the indicated motifs. Expression of HA-MITF WT and mutants induced at 0 or 20 ng doxycycline. Colored line indicates median, and black line indicates mean. See also Figure S4 .

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Genome Wide, Mutagenesis, Derivative Assay, ChIP-sequencing, Expressing, Whisker Assay

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: Live-Cell Single-Molecule Tracking (SMT) of HALO-Tagged MITF (A) HALO-tagged MITF expression vectors. NLS indicates the nuclear localization sequence. Δbasic lacks residues required for DNA binding. (B) Exemplary frames of SMT movies using WT and mutant HALO-tagged MITF, collected at 100 fps (see also , , , and ). Scale bar, 5 μm. Labeling with 100 pM Halotag JF 594 allows particle densities in the range of a few molecules per frame. (C) SMT movies were tracked to generate a distribution of single-molecule displacements between consecutive frames that was fit with a three-component model (one immobile component and two diffusing components) to provide quantitative estimates for WT MITF and mutants shown in (D) and (E). Cmp, component. (D) Quantitative estimates derived from SMT using WT and mutant HALO-tagged MITF for the fraction of molecules in each state. Error bars indicate SD. (E) Quantitative estimates of the diffusion coefficients of free molecules. For MITF WT, Δ basic, K243Q, and K243R, respectively, N c e l l s = 20 , 6 , 15 , 15 ; N d i s p l a c e m e n t s = 17802 , 2684 , 16422 , 12999 . Error bars indicate SD. (F) Summary derived from the SMT analysis of proportion of MITF calculated to bind high- versus low-affinity sites. (G) Electrophoretic mobility shift assay (EMSA) using bacterially expressed and purified WT and mutant MITF DNA-binding domains, a 30-bp TCACGTGA-motif-containing probe, and competition with 4-fold dilutions of SSD (10 μg to 2.3 fg). Bound DNA is shown. Probe was in excess in all reactions. (H) EMSA as in (G) with competition by indicated competitor oligonucleotides at 3, 10, and 30 ng. Bound DNA is shown. See also and and , , , and .

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Expressing, Sequencing, Binding Assay, Mutagenesis, Labeling, Derivative Assay, Diffusion-based Assay, Electrophoretic Mobility Shift Assay, Purification