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: The Journal of Clinical Investigation

Article Title: Natural killer cells activated through NKG2D mediate lung ischemia-reperfusion injury

doi: 10.1172/JCI137047

Figure Lengend Snippet: To determine if NK cells expressed tissue-resident markers, lungs and peripheral blood were collected from C57BL/6 mice (n = 4) following HC (n = 2) or sham (n = 2) procedures. Lung and blood NK cells were phenotypes by spectral flow cytometry and populations were identified based on expression of key markers. (A and B) Cell populations appeared to cluster by tissue origin, but not treatment condition. (C) L-selectin (CD62L) was decreased on lung NK cells relative to blood. Pulmonary NK cells displayed a more activated phenotype with increased CD16 (D), KLRG1 (E), Ly6c (F), and CD11b (G) and decreased CD27 (H) and DNAM1 (I). There were trends for decreased TRAIL (J) and NKG2A/C/E (K) in lung relative to blood NK cells. There was a positive correlation between KLRG1 and DNAM1 in the lung compared with the blood. Box and whisker plots show individual data points bound by boxes at 25th and 75th percentiles and medians depicted with bisecting lines. Differences were assessed with paired Student’s t tests and statistically significant correlations (P < 0.05) between lung and blood NK cells are shown as Pearson’s coefficient (r).

Article Snippet: NK cell subsets were identified using: Allophycocyanin–conjugated (APC-conjugated) anti-NKG2A(clone 131411, R&D Systems, catalog FAB1059A), R-phycoerythrin–conjugated (PE-conjugated) anti-KIR2D (clone NKVFS1, Miltenyi Biotec, catalog 130-092-688), PE-conjugated anti-KIR3D (clone Z27.3.7, Beckman Coulter, catalog IM3292), peridinin-chlorophyll-protein complex–conjugated (PerCP-conjugated) anti-KIR3DL1 (clone DX9, BioLegend, catalog 312718), PE-Cy7–conjugated anti-CD56 (clone HCD56, BioLegend, catalog 318318), Alexa Fluor 700–conjugated anti-CD45 (clone HI30, BioLegend, catalog 304024), and allophycocyanin-cyanine 7–conjugated (APC-Cy7–conjugated) anti-CD3 (clone SK7, BioLegend, catalog 344818).

Techniques: Flow Cytometry, Expressing, Whisker Assay

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