Journal: Brain, behavior, and immunity

Article Title: Early life stress impairs synaptic pruning in the developing hippocampus

doi: 10.1016/j.bbi.2022.09.014

Figure Lengend Snippet: ELS reduces expression of TREM2 and CD11b while increasing CX3CR1 in P17 microglia. (A) Scatter plots for quantifying Trem2 positive microglia in CTL, LB and UPS. (B) Percentage of TREM2-positive microglia. Median fluorescence intensity (MFI) plots and surface expression of CD11b (C-D), CD45 (E-F) and CX3CR1 (G-H). CTL-males: n = 16, CTL-females: n = 14, LB males: n = 10, LB-females: n = 9, UPS-males: n = 12, UPS-females: n = 16. Error bars represent mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Microglia were isolated from the hippocampus as described above and blocked with rat- anti CD16/CD32 Fc antibodies 1:100 (Cat# 553142, BD Biosciences) and then stained with rat-anti CD11b-PeCy7 1:100 (Cat# 25-0112-82, ebiosciences), rat-anti CD45-PeCPCy5.5 antibodies 1:100 (Cat# 45-0451-82, ebiosciences), APC-rat anti Trem2 antibodies 1:10 (Cat# FAB17291A R & D systems), or APC-mouse anti CX3CR1 antibodies 1:100 (Cat# 149008) for 25 min on ice.

Techniques: Expressing, Fluorescence

Journal: Brain, behavior, and immunity

Article Title: Early life stress impairs synaptic pruning in the developing hippocampus

doi: 10.1016/j.bbi.2022.09.014

Figure Lengend Snippet: Trem2 is essential for normal phagocytic activity. (A-B) Effects of Trem2 genotype (WT, Hets, Ko) on ex vivo phagocytic activity in microglia isolated from the hippocampus of 17-day old pups (n = 10–17 pups per group, 50 % females). (C) Representative confocal images (top row) and Imaris models (middle and lower rows) of microglia from Trem2 wildtype (WT), heterozygous (Hets) and knockout P17 littermates. Staining for Iba1 (green), CD68 (blue), and PSD95 (red). Middle row: reconstruction of Iba1 & CD68 staining. Lower row: reconstruction of CD68 and PSD95 staining inside microglia. Effects of Trem2 genotype on microglial volume (D), CD68 volume inside microglia (E), number of PSD95 puncta in microglia (F) and PSD95 puncta inside CD68 (G), n = 5 cells per mouse and 4–5 mice per group, 50 % females). Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Microglia were isolated from the hippocampus as described above and blocked with rat- anti CD16/CD32 Fc antibodies 1:100 (Cat# 553142, BD Biosciences) and then stained with rat-anti CD11b-PeCy7 1:100 (Cat# 25-0112-82, ebiosciences), rat-anti CD45-PeCPCy5.5 antibodies 1:100 (Cat# 45-0451-82, ebiosciences), APC-rat anti Trem2 antibodies 1:10 (Cat# FAB17291A R & D systems), or APC-mouse anti CX3CR1 antibodies 1:100 (Cat# 149008) for 25 min on ice.

Techniques: Activity Assay, Ex Vivo, Isolation, Knock-Out, Staining

Journal: Stem Cell Reports

Article Title: Resistance to Taxanes in Triple-Negative Breast Cancer Associates with the Dynamics of a CD49f+ Tumor-Initiating Population

doi: 10.1016/j.stemcr.2017.03.026

Figure Lengend Snippet: The CD49f+ Population Is Enriched after Acquisition of Resistance to Docetaxel (A) Frequency of indicated markers within the H2Kd– population in IDB-01 and IDB-02, sensitive and resistant tumors analyzed by flow cytometry at passage 3, at least 5 days after the last docetaxel treatment. Total number of tumors analyzed (n) mean values, SDs and t test p values are shown. ∗ 0.01 < p < 0.05; ∗∗ 0.001 < p < 0.01. (B) Box and whiskers (min to max) graph showing expression levels of CD49f and EpCAM mRNA relative to PPiA in additional sensitive and resistant tumors measured by qRT-PCR. Determinations were done in triplicate and means are used. t test p values for significant differences are shown. ∗∗ 0.001 < p < 0.01. (C) Box and whiskers (min to max) graph showing association of CD49f and EpCAM with chemotherapy response in 74 patients with basal-like breast cancer EORTC ( Bonnefoi et al., 2011 ). Response was measured as pathological complete response (pCR) or residual disease (RD). t test p values are shown. (D) Kaplan-Meier analysis of overall survival of ER-tumors all treated with chemotherapy using CD49f and EpCAM mRNA expression in the clinical dataset (GSE16446) from the TOP TRIAL ( Desmedt et al., 2011 ). See also and .

Article Snippet: Then they were labeled with antibodies against CD24-PE (555428), CD44-APC (559942), EpCAM-FITC (347197), CD10-PECy5 (555376), and CD49f-A647 (562473) (all from BD Pharmingen), CD133/1-PE (130-098-826 from Miltenyi Biotec), and CD49f-APC (FAB13501A from R&D Systems).

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: Stem Cell Reports

Article Title: Resistance to Taxanes in Triple-Negative Breast Cancer Associates with the Dynamics of a CD49f+ Tumor-Initiating Population

doi: 10.1016/j.stemcr.2017.03.026

Figure Lengend Snippet: CD49f Expression Increases in Residual Disease of Most TNBC PDX Tumors after Treatment with Docetaxel, but Not on Resistant Tumors (A) Scheme of short-term docetaxel treatment and CD49f mRNA expression levels in sensitive tumors from IDB-01S and IDB-02S after short-term treatment with docetaxel (DTX) and in untreated controls (CT). Each dot represents one tumor. ∗ 0.01 < p < 0.05; ∗∗ 0.001 < p < 0.01. (B) Frequency of CD49f+ cells within H2Kd– in IDB-01S tumors after two to three doses of docetaxel and in IDB-01R tumors that have been treated with docetaxel (at least 5 days after last treatment) or growing in the absence of docetaxel for two and five passages. ∗ 0.01 < p < 0.05; ∗∗ 0.001 < p < 0.01. (C and D) Docetaxel-sensitive tumors (C) and docetaxel-resistant tumors (D). Top panels: tumor size of the indicated PDX tumors treated with docetaxel (20 mg/kg, arrows) and corresponding controls relative to the size at the first day of treatment. n = total number of tumors. ∗∗∗∗ p < 0.0001. Bottom panels: CD49f mRNA expression levels in PDX tumors after short-term treatment with docetaxel and in untreated controls. Each dot represents one tumor. ∗ 0.01 < p < 0.05; ∗∗ 0.001 < p < 0.01; ∗∗∗ 0.001 < p < 0.0001. (A–D) Mean values, SEM, and t test p values are shown in all cases. See also Figure S5 .

Article Snippet: Then they were labeled with antibodies against CD24-PE (555428), CD44-APC (559942), EpCAM-FITC (347197), CD10-PECy5 (555376), and CD49f-A647 (562473) (all from BD Pharmingen), CD133/1-PE (130-098-826 from Miltenyi Biotec), and CD49f-APC (FAB13501A from R&D Systems).

Techniques: Expressing

Journal: Stem Cell Reports

Article Title: Resistance to Taxanes in Triple-Negative Breast Cancer Associates with the Dynamics of a CD49f+ Tumor-Initiating Population

doi: 10.1016/j.stemcr.2017.03.026

Figure Lengend Snippet: CD49f Expression Increases in Surviving TNBC Cells after Treatment with Docetaxel (A and B) Top panels: percentage of surviving cells treated with docetaxel for 72 h (A) or 8 h (B). Bottom panels: CD49f mRNA expression levels in the indicated TNBC cell lines treated with docetaxel relative to untreated controls. ∗ 0.01 < p < 0.05; ∗∗ 0.001 < p < 0.01; ∗∗∗ 0.001 < p < 0.0001. (C and D) CD49f mRNA expression levels (C) and CD49f protein expression measured by flow cytometry (D) in cells stably infected with two independent shCD49f knockdown constructs and control vector (pGIPZ). (E) Percentage of surviving shCD49f-infected and control pGIPZ-infected cells treated with indicated doses of docetaxel for 72 hr. RT-PCR Determinations were done in triplicate and means are used in the calculations. Mean values of three independent experiments, SEM, and t test p values for the higher concentrations are shown. See also Figure S5 .

Article Snippet: Then they were labeled with antibodies against CD24-PE (555428), CD44-APC (559942), EpCAM-FITC (347197), CD10-PECy5 (555376), and CD49f-A647 (562473) (all from BD Pharmingen), CD133/1-PE (130-098-826 from Miltenyi Biotec), and CD49f-APC (FAB13501A from R&D Systems).

Techniques: Expressing, Flow Cytometry, Stable Transfection, Infection, Knockdown, Construct, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Stem Cell Reports

Article Title: Resistance to Taxanes in Triple-Negative Breast Cancer Associates with the Dynamics of a CD49f+ Tumor-Initiating Population

doi: 10.1016/j.stemcr.2017.03.026

Figure Lengend Snippet: CD49f+ Population Is Enriched in Tumor-Initiating Cells with Increased Resistance to Docetaxel (A) Scheme of functional experiments. (B and E) Table showing limiting dilution assay of CD49f+/hi and CD49f− tumor cells from IDB-01 (B) and IDB-02 (E) cells. Tumor-initiating cell frequency (with confidence intervals) for each group was calculated by ELDA; chi-square values and associated probabilities are shown. (C and G) Frequency of CD49f+ cells in tumors derived from indicated cells. Mean values, SEM, and significant t test p values are shown. ∗∗ 0.001 < p < 0.01; ∗∗∗ 0.001 < p < 0.0001. (D and H) Kinetics of tumor growth during docetaxel treatment in tumors derived from indicated cells. Mean values, SEM, and t test p values are shown. ∗∗ 0.001 < p < 0.01; ∗∗∗∗ p <0.0001. (F) Latency of tumors derived from the injection of the indicated number of IDB-02S-CD49f+/hi and 02S-CD49f− tumor cells. Mean values, SEM and significant t test p values are shown. ∗∗ 0.001 < p < 0.01. (I) Unsupervised analysis of all CD49f sorted samples from IDB-01S and -01R tumors using 105 breast cancer-related genes. The type of sample and tumor are shown below the array tree. Each square represents the relative transcript abundance. See also Figure S6 .

Article Snippet: Then they were labeled with antibodies against CD24-PE (555428), CD44-APC (559942), EpCAM-FITC (347197), CD10-PECy5 (555376), and CD49f-A647 (562473) (all from BD Pharmingen), CD133/1-PE (130-098-826 from Miltenyi Biotec), and CD49f-APC (FAB13501A from R&D Systems).

Techniques: Functional Assay, Limiting Dilution Assay, Derivative Assay, Injection

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: The NLRP3 inflammasome pathway is upregulated in fibroblasts during mammary carcinogenesis. a Scheme of fibroblast isolation procedure. b Principal component analysis (PCA) of samples included in the microarray transcriptome analysis. Each focal point represents a cohort of mice. Normal n = 6 mice/cohort, Hyperplasia n = 6 mice/cohort, Carcinoma n = 4 mice/cohort, advanced carcinoma (A. Carcinoma) n = 3 mice/cohort. c Venn diagrams depicting overlap of differentially expressed genes in fibroblasts isolated from distinct stages of MMTV-PyMT mammary carcinoma. d Pro-inflammatory genes upregulated in CAFs isolated from MMTV-PyMT tumours. Inflammasome-related genes are highlighted. e Heat map of Nlrp3/Il1b pathway-related genes. f qRT-PCR analysis of Nlrp3/Il1b pathway related genes in fibroblasts isolated by FACS (PDGFRα + CD45 − EpCAM − ) from MMTV-PyMT mice or normal mammary glands. Data are presented as mean ± s.d of technical repeats; One-way analysis of variance followed by Tukey’s test. * p < 0.05, ** p < 0.005, *** p < 0.0005, n = pools of 3 mice/group. Results are representative of three independent biological experiments. g Representative IHC staining of NLRP3 in PyMT tumours or in normal mammary glands. Arrows indicate fibroblasts. Scale bar, 25 µm. h Quantification of staining shown in g . Multiple fields of 3 mice/group were analysed for the percentage of NLRP3 expressing fibroblasts out of total fibroblasts. Data are presented as mean ± s.e.m; Mann–Whitney test. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Isolation, Microarray, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing, MANN-WHITNEY

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: The NLRP3 inflammasome pathway is up-regulated in CAFs in human breast tumours. a – d Expression levels of Nlrp3/Il1b pathway-related genes in tumour-associated stroma of breast cancer patients with infiltrating ductal carcinoma (IDC) staged 1/2 vs. 3 ( n = 53) or in normal breast stroma ( n = 6). Data was obtained from NCBI GEO (Dataset accession number GSE 9014) and is presented as mean ± s.e.m.; One-way analysis of variance test followed by Tukey’s multiple comparisons test. e – g Analysis of NLRP3 expression in fibroblasts in human IDC or in normal breast tissue sections. Images were obtained from the Human Protein Atlas. e Representative images, arrows in insets indicate fibroblasts. f Quantification of the percentage of NLRP3-expressing fibroblasts out of total fibroblasts for each sample. n = 2 samples for normal and 8 samples for IDC, error bars represent s.e.m; Welch’s t -test. g Quantification of staining intensity for NLRP3 in fibroblasts shown in e . h – j Staining for IL-1β in benign or malignant (DCIS/IDC) tissue sections from human breast cancer patients. h Representative images of stained benign and IDC breast tissue sections. Arrows indicate fibroblasts. Scale bar, 50 µm. i , j Quantification of the abundance i and staining intensity j of labelled fibroblasts, n = 73 (benign: n = 31, DCIS: n = 16, IDC: n = 26); Mann–Whitney test ( i ) or Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( j ). Data are presented as mean ± s.e.m; Mann–Whitney test. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: Fibroblasts function as DAMP sensors via NLRP3 inflammasome signalling. a – e Primary FVB/n NMFs were incubated for 24 h in control medium or in medium containing one of the following DAMPs: ATP 1 mM, H 2 O 2 300 μM, MSU 5 μg/mL, 5% necrotic fluid (extracted from late-stage PyMT tumours). Nlrp3 a and Il1b b expression in fibroblasts were analysed by qRT-PCR. Data are presented as mean ± s.d.; One-way analysis of variance test followed by Dunnett’s multiple comparisons test. Representative of three independent experiments. c Caspase-1 processing was assessed by western blot of cell lysates with anti-Caspase-1. β-actin was utilised as a loading control. The samples shown are derived from the same experiment. Data are representative of three independent experiments. d ELISA quantification of IL-1β secretion. Data are presented as mean ± s.d. of biological replicates ( n = 4 per group); Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Representative of three independent experiments. e Primary FVB/n NMFs were incubated for 24 h with 5% necrotic fluid or control medium and expression of selected genes was analysed using qRT-PCR. Data are presented as mean ± s.d. of biological replicates ( n = 3 per group); Welch’s t -test. Representative of two independent experiments. f Schematic illustration of cutaneous wound assay. Cutaneous wounds were generated in 8 weeks old FVB/n mice using the dermal punch method. Wounded or control skin fibroblasts were isolated by FACS as PDGFRα + CD45 − EpCAM − cells. g qRT-PCR analysis of the expression of selected genes in wound-derived fibroblasts as compared to their expression in fibroblasts from normal skin. n = pool of 8 mice per group. Data are presented as mean ± s.d. of biological repeats; Welch’s t -test. Representative of three independent biological experiments. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Generated, Isolation

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: CAF-derived NLRP3 inflammasome signalling facilitates tumour growth. a Scheme of experiments analysed in b – h . shNlrp3 AT3 cells were orthotopically co-injected with WT ( Nlrp3 +/+ ) or with Nlrp3 −/− NMFs (C57BL/6) into C57BL/6 mice. b Growth curves of AT3 tumours. n = 7 mice per group. c Tumour weights at termination of experiment. n = 6 and 7 mice per group (WT and Nlrp3 −/− , respectively). d Flow cytometry analysis of Gr1 + cell infiltration into AT3 tumours co-injected with WT NMFs ( n = 4) or with Nlrp3 −/− NMFs ( n = 3). Data presented are percentage of CD45 + cells, normalised to WT. e Representative images of staining with anti-Gr1 antibody in AT3 tumours. Scale bar, 100 μm. f Quantification of staining shown in d . Three fields of 3 mice/group were analysed; Data are presented as mean Gr1 + cells/field ± s.d; Mann–Whitney test. g , h Flow cytometry analysis of CD11b + Ly6C high Ly6G − and CD11b + Ly6G + Ly6C − cell infiltration into AT3 tumours co-injected with WT NMFs ( n = 4) or with Nlrp3 −/− NMFs ( n = 3). Data presented are percentage of CD45 + cells, normalised to WT. i Scheme of experiments analysed in j – r . Met-1 cells were orthotopically co-injected with NMFs depleted of Nlrp3 or Il1b expression (shNlrp3, and shIl1b), or with control NMFs (shScramble). j Growth curve and k weight at endpoint of Met-1 tumours. n = 5, 10, 20, 19 tumours (TCs only, shScramble, shNlrp3, and shIl1b, respectively). l – n Flow cytometry analysis of Gr1 + cell infiltration into Met-1 tumours. n = 7, 14, and 16 per group (shScramble, shNlrp3, and shIl1b, respectively). Data presented are percentage of CD45 + CD11b + cells, normalised to shScramble. o – r Met-1 tumours were analysed for the expression of selected genes using qRT-PCR. n = 6, 7, and 5–6 tumours per group (shScramble, shNlrp3, and shIl1b, respectively). Data are presented as fold change from shScramble. In b – d , g , h , j , k , data are presented as mean ± s.e.m; Welch’s t -test, * p < 0.05, ** p < 0.005. In j – r data are presented as mean ± s.e.m; One-way analysis of variance test followed by Tukey’s multiple comparisons test. Data are representative of three independent biological experiments. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Injection, Flow Cytometry, Staining, MANN-WHITNEY, Expressing, Quantitative RT-PCR

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: Summary. Activation of the NLRP3 inflammasome in cancer-associated fibroblasts links tissue damage with tumour-promoting inflammation in breast cancer progression and metastasis. Figure preparation partially involved elements from the Servier Medical Art. Figure preparation involved using elements from the Servier Medical Art and somersault18:24 (somersault1824.com)

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Activation Assay