Journal: bioRxiv

Article Title: CECR2 Drives Breast Cancer Metastasis by Suppressing Macrophage Inflammatory Responses

doi: 10.1101/2020.09.10.291799

Figure Lengend Snippet: ( A-B ) Gene set enrichment analysis comparing transcriptomes of CECR2 knockout (CECR2 sg1 and CECR2 sg2) with control LM2 cells. Venn diagram ( A ) showing the number of shared downregulated hallmark pathways ( B ). ( C ) RT-qPCR analysis of CSF1, CSF2 and CXCL1 in control and CECR2 knockout LM2 cells treated with 20 ng/ml TNF-α for 3 hours. ( D ) RT-qPCR analysis of Csf1, Csf2 and Cxcl1 in control 4T1, cecr2 knockout 4T1 and cecr2 knockout 4T1 with CECR2 reconstituted expression after treatment with 20 ng/ml TNF-α for 3 hours. ( E-F ) Western blot analysis of cell lysates (input) and immunoprecipitates (IP) from 4T1 ( E ) and LM2 ( F ) cells stimulated with 20 ng/ml TNF-α for 0.5 hour with the indicated antibodies. ( G-H ) ChIP-qPCR analyses with the indicated antibodies of the CSF1 promoter in control, CECR2 knockout (CECR2 sg1 and CECR2 sg2) ( G ), and RELA knockout (RELA sg) ( H ) LM2 cells stimulated with 20 ng/ml TNF-α for 0.5 hour. * p < 0.05, ** p < 0.01, *** p < 0.001. Representative data from triplicate experiments are shown, and error bars represent SEM.

Article Snippet: The following antibodies were obtained commercially: rabbit anti-CECR2 (HPA002943), mouse anti-FLAG (M2, F1804), and mouse anti-tubulin (T5168) (Sigma, St. Louis, MO); mouse anti-CECR2 (C3, sc-514878), mouse anti-CSF1 (D4, sc-365779), mouse anti-NF-κB p50 (E-10, sc-8414) (Santa Cruz, Dallas, TX); rabbit anti-NF-κB p65 (D14E12, #8242), rabbit anti-NF-κB2 p100/p52 (#4882), rabbit anti-RelB (C1E4, #4922), rabbit anti-c-Rel (D4Y6M, #12707), mouse anti-GAPDH (D4C6R, #97166) (Cell Signaling Technology, Danvers, MA); rabbit anti-H3(ab1791), mouse anti-RNA pol II (8WG16, ab817) (Abcam, Cambridge, UK); rabbit anti-T7 (AB3790) and rabbit anti-H3K9/18Ac (07-593) (Millipore sigma, Burlington, MA).

Techniques: Knock-Out, Control, Quantitative RT-PCR, Expressing, Western Blot, ChIP-qPCR

Journal: bioRxiv

Article Title: CECR2 Drives Breast Cancer Metastasis by Suppressing Macrophage Inflammatory Responses

doi: 10.1101/2020.09.10.291799

Figure Lengend Snippet: ( A ) Western blot analysis of cell lysates (Input) and anti-FLAG immunoprecipitates (IP) from HEK293T cells transfected with the indicated combination of vectors expressing FLAG-CECR2, K310R mutated RELA and WT RELA. ( B ) Western blot analysis of cell lysates (Input) and anti-FLAG immunoprecipitates (IP) from HEK293T cells transfected with the indicated combination of vectors expressing WT FLAG-CECR2, FLAG-CECR2 mutant with bromodomain deletion (ΔBRD) and T7-RELA. ( C ) Western blot analysis of cell lysates (input) and anti-RELA immunoprecipitates (IP) from LM2 cells pretreated with control DMSO, CECR2 inhibitor 1 μM NVS-CECR2-1 or 1 μM GNE-886 for 2 days, and then stimulated with 20 ng/ml TNF-α for 0.5 hour. ( D-E ) RT-qPCR analyses of CSF1, CSF2 and CXCL1 in LM2 cells pretreated with the indicated concentration of NVS-CECR2-1 ( D ) or GNE-886 ( E ) for 2 days and then stimulated with 20 ng/ml TNF-α for 3 hours. ( F) Scratch migration assays comparing the closure of wound healing distance in LM2 cells treated with DMSO, 1 μM NVS-CECR2-1 or 1 μM GNE-886 for 2 days. ( G ) Transwell invasion assays comparing LM2 cells treated with DMSO, 1 μM NVS-CECR2-1 or 1 μM GNE-886 for 2 days. * p < 0.05, ** p < 0.01, *** p < 0.001. Representative data from triplicate experiments are shown, and error bars represent SEM.

Article Snippet: The following antibodies were obtained commercially: rabbit anti-CECR2 (HPA002943), mouse anti-FLAG (M2, F1804), and mouse anti-tubulin (T5168) (Sigma, St. Louis, MO); mouse anti-CECR2 (C3, sc-514878), mouse anti-CSF1 (D4, sc-365779), mouse anti-NF-κB p50 (E-10, sc-8414) (Santa Cruz, Dallas, TX); rabbit anti-NF-κB p65 (D14E12, #8242), rabbit anti-NF-κB2 p100/p52 (#4882), rabbit anti-RelB (C1E4, #4922), rabbit anti-c-Rel (D4Y6M, #12707), mouse anti-GAPDH (D4C6R, #97166) (Cell Signaling Technology, Danvers, MA); rabbit anti-H3(ab1791), mouse anti-RNA pol II (8WG16, ab817) (Abcam, Cambridge, UK); rabbit anti-T7 (AB3790) and rabbit anti-H3K9/18Ac (07-593) (Millipore sigma, Burlington, MA).

Techniques: Western Blot, Transfection, Expressing, Mutagenesis, Control, Quantitative RT-PCR, Concentration Assay, Migration

Journal: bioRxiv

Article Title: CECR2 Drives Breast Cancer Metastasis by Suppressing Macrophage Inflammatory Responses

doi: 10.1101/2020.09.10.291799

Figure Lengend Snippet: ( A-B ) BALB/c wild type mice were injected with control 4T1, Cecr2 knockout (sg1) 4T1 cells, or Cecr2 knockout 4T1 cells with CSF1 overexpression (n=8 for all the groups) through tail vein. Metastatic lesions in the lungs at week 3 after tumor cell injection were stained by India ink. Shown are representative images ( A ) and quantification of metastases in the lungs ( B ). ( C-D ) H&E staining of the lungs from mice in (A) at week 3. Shown are representative images ( C ) and quantification of tumor areas in the lungs ( D ). Scale bars: 200 μm. ( E-G ) Flow cytometry analysis of lung lesions from BALB/c wild type mice injected with control 4T1, Cecr2 knockout (sg1) 4T1 cells, or Cecr2 knockout 4T1 cells with CSF1 overexpression (n=8 for (E-F), n=3 for (G)) through tail vein at week 3. Shown are quantification of the percentages of total macrophages (CD45+F4/80+) ( E ), M2 macrophages (CD45+F4/80+CD206+) ( F ) and Granzyme B (GZMB)+ CD8+ T cells (CD45+CD8+GZMB+) ( G ). GZMB, Granzyme B. * p <0.05; *** p <0.001, n.s. not significant. Representative data from triplicate experiments are shown, and error bars represent SEM. ( H ) Schematic illustration of intraperitoneal injection (i.p.) of NVS-CECR2-1 (10 μg/injection/mouse) or equal volume of PBS every other day for 28 days one day after tail vein injection of 4T1 cells (1×105/mouse) in BALB/c mice. All mice were sacrificed on day 35 to collect lungs and H&E staining were performed. ( I-K ) Representative H&E staining ( I ), quantification of total tumor lesions per lung ( J ) and percentage of tumor area per lung ( K ) of lungs from BALB/c mice in ( H ). ( L ) Graphical model of CECR2 promotes breast cancer metastasis by binding to RELA through its bromodomain (BD) and activating NF-κB response genes including CSF1 to modulate the immune suppressive microenvironment at the metastatic site.

Article Snippet: The following antibodies were obtained commercially: rabbit anti-CECR2 (HPA002943), mouse anti-FLAG (M2, F1804), and mouse anti-tubulin (T5168) (Sigma, St. Louis, MO); mouse anti-CECR2 (C3, sc-514878), mouse anti-CSF1 (D4, sc-365779), mouse anti-NF-κB p50 (E-10, sc-8414) (Santa Cruz, Dallas, TX); rabbit anti-NF-κB p65 (D14E12, #8242), rabbit anti-NF-κB2 p100/p52 (#4882), rabbit anti-RelB (C1E4, #4922), rabbit anti-c-Rel (D4Y6M, #12707), mouse anti-GAPDH (D4C6R, #97166) (Cell Signaling Technology, Danvers, MA); rabbit anti-H3(ab1791), mouse anti-RNA pol II (8WG16, ab817) (Abcam, Cambridge, UK); rabbit anti-T7 (AB3790) and rabbit anti-H3K9/18Ac (07-593) (Millipore sigma, Burlington, MA).

Techniques: Injection, Control, Knock-Out, Over Expression, Staining, Flow Cytometry, Binding Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Impaired SNF2L Chromatin Remodeling Prolongs Accessibility at Promoters Enriched for Fos/Jun Binding Sites and Delays Granule Neuron Differentiation

doi: 10.3389/fnmol.2021.680280

Figure Lengend Snippet: Generation of GNP cultures from the Ex6DEL mice. (A) Schematic diagram of the procedure for generating GNP and granule neuron cultures. (B) Representative images of WT cultures 1 DIV (top image) and 3 DIV (bottom image) stained with neurofilament-200 (red). Scale bar, 25 μM. (C) Protein extracts of primary GNP cultures at I and 3 DIV were immunoblotted for the ISWI proteins (Snf2h, Snf2l), neuronal (Tuj, NeuN) and proliferation (Ki67) markers. Vinculin was used as a loading control. (D) Immunoblot (top panel), Smarca1 genotyping (middle panel), and RNAseq analysis (bottom panel) from WT and Ex6DEL GNP cultures confirmed the loss of exon 6 in the GNP cultures isolated from Ex6DEL mice. The red rectangle outlines the absence of RNAseq reads corresponding to the position of exon 6. (E) Phase contrast images of GNP cultures from WT and Ex6DEL mice. Scale bar, 50 μM.

Article Snippet: Membranes were blocked (45 min, room temperature) with 5% skim milk in TBST and incubated (4°C, overnight) in primary antibody [rabbit anti-Snf2l (1:2000, Abcam, ab37003); rabbit anti-Snf2h (1:2000, Abcam, ab72499); rabbit anti-vinculin (1:2000, Abcam, ab129002); rabbit anti-Ki67 (1:2000, Abcam, ab16667); mouse anti-NeuN (1:2000, Millipore, MAB377); mouse anti-Tuj1 (1:2000, Stem Cell Technologies, 01409); rabbit anti-CECR2 (1:1000; gift from Dr. Heather McDermid, uAlberta); rabbit anti-ERK (1:2000, Santa Cruz, sc154); or mouse anti-pERK (1:500, Santa Cruz, sc7383)].

Techniques: Staining, Western Blot, Isolation