Journal: British Journal of Cancer

Article Title: Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

doi: 10.1038/s41416-020-0804-z

Figure Lengend Snippet: a Proteome profiler Human XL Cytokine Array (ARY022, R&D Systems, MN, USA) probed with serum-free-conditioned medium (SF-CM) of C33a-FL-CA IX or C33a-NS-CA IX cells incubated under normoxic (NO) and hypoxic (HY) conditions for 48 h. Spot doublets of corresponding proteins with differential intensities are framed and indicated by arrows. Medium pooled from five independent experiments was used for the array. Spot intensities representing b pro-oncogenic basigin, and c anti-angiogenic thrombospondin-1 were analysed in culture medium samples of C33a-FL-CA IX versus C33a-NS-CA IX cells. Abundance of the proteins was assessed according to spot intensity using densitometry. Data are shown as mean ± SD. d Validation of the differential levels of basigin and thrombospondin-1, respectively, by Western blotting analysis of medium precipitates (MP) and cell lysates (CL) of normoxic (NO) and hypoxic (HY) cells. (* P < 0.05, ** P < 0.01, *** P < 0.005).

Article Snippet: PPA analyses were performed using the Human XL Cytokine Array Kit and Human Soluble Receptor Array Non-Hematopoietic panel kit (R&D Systems, MN, USA).

Techniques: Incubation, Biomarker Discovery, Western Blot

Journal: British Journal of Cancer

Article Title: Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

doi: 10.1038/s41416-020-0804-z

Figure Lengend Snippet: Analysis of the differential expression of a IGFBP-2 and b IGFBP-3 in SF-CM samples from C33a-FL-CA IX and C33a-NS-CA IX incubated under normoxic (NO) and hypoxic (HY) conditions for 48 h and evaluated by Proteome Profiler Human XL Cytokine Array (ARY022, R&D Systems, MN, USA). Abundance of the proteins was assessed according to spot intensity using densitometry. Data are shown as mean ± SD of five independent samples. c Western blotting analysis of CA IX, IGFBP-2 and IGFBP-3 protein levels in medium precipitates (MP) of C33a-FL-CA IX and C33a-NS-CA IX cells. Impairment of shedding in NS-CA IX is associated with increased IGFBP-2 and reduced IGFBP-3 secretion to culture medium. Quantitative PCR analysis of relative mRNA levels of d IGFBP-2, e IGFBP-3 and f IGF1 in C33a-FL-CA IX versus C33a-NS-CA IX cells normalised to β-actin mRNA. (*** P < 0.005, ns non-significant).

Article Snippet: PPA analyses were performed using the Human XL Cytokine Array Kit and Human Soluble Receptor Array Non-Hematopoietic panel kit (R&D Systems, MN, USA).

Techniques: Quantitative Proteomics, Incubation, Western Blot, Real-time Polymerase Chain Reaction

Journal: International journal of oncology

Article Title: Oct4 suppresses IR‑induced premature senescence in breast cancer cells through STAT3- and NF‑κB-mediated IL‑24 production.

doi: 10.3892/ijo.2018.4391

Figure Lengend Snippet: Figure 8. Function of IL‑24 in Oct4‑mediated suppression of IR‑induced senescence. (A) Cytokine array of MCF7 cells. RT‑PCR analysis of MCF7 cells (B) cultured as indicated with the indicated primers. (C) Western blot analysis of adenoviral‑Luc- and adenoviral‑Oct4‑transduced MCF7 cells. RT‑PCR analysis of MCF7 cells (D) exposed to 4 Gy of IR, (E) treated with STAT3 inhibitor WP1066 (5 µM), (F) transfected with siRNA-targeting STAT3 or (G) treated with NF‑κB inhibitor Bay 11-7082 (10 and 20 µM) following transduction with adenoviral‑Luc or adenoviral‑Oct4. (H) Western blot analysis of adenoviral‑Luc- and adenoviral‑Oct4‑transduced MCF7 cells pretreated with Bay 11-7082 (10 µM) with the indicated antibodies. IL, interleukin; Oct4, octamer-binding transcription factor 4; RT‑PCR, reverse transcription-polymerase chain reaction; IR, ionizing radiation; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3; NF‑κB, nuclear factor κB; IκB, inhibitor of NF‑κB; SDF‑1α, stromal cell-derived factor 1α; GDF15, growth differentiation factor 15; MCP‑1, monocyte chemoattractant protein 1; CCL, CC chemokine ligand; PAI‑1, plasminogen activator 1; Rantes, regulated on activation, normal T-cell expressed and secreted; IP‑10, interferon γ-induced protein 10; CXCL10, CXC cytokine ligand 10; DKK1, dickkopf-related protein 10; msp, mammosphere; mono, monolayer- cultured; IRR, IR‑resistant; Luc, luciferase; siSTAT3, small interfering RNA against STAT3; sicon, control small interfering RNA; p-, phosphorylated.

Article Snippet: Cytokine array-base analysis was performed using a Human Cytokine Array kit (cat. no. ARY022; R&D Systems, Inc.), according to the manufacturer's protocol.

Techniques: Cell Culture, Western Blot, Transfection, Transduction, Binding Assay, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA, Derivative Assay, Activation Assay, Luciferase, Control

Journal: Biology

Article Title: Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium.

doi: 10.3390/biology11070980

Figure Lengend Snippet: Figure 6. Profiling of cytokines expressed by secretomes of irradiated or non-irradiated fibroblasts obtained with Proteome Profiler Human XL cytokine Array Kit (R&D) (A) Representative image of dot blot, (B) graph showing signal density of dot blot for each protein normalized to positive spot pixels.

Article Snippet: Profili cytokines expres ed by secretomes of irradiated or non-irradi ted fibro lasts obtained with Proteome Profile Human XL cytokine Array Kit (R&D) (A) Representative image of dot blot, (B) graph showing signal density of d t blot f r each protein normalized to positive spot pixels.

Techniques: Irradiation, Dot Blot

Journal: Biology

Article Title: Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium.

doi: 10.3390/biology11070980

Figure Lengend Snippet: Figure 8. Profiling of cytokines expressed by secretomes of irradiated untreated fibroblast or precon- ditioned with MSC-EV or MSC-CM obtained with Proteome Profiler Human XL cytokine Array Kit (R&D). (A) Representative image of dot blot; (B) graph showing signal density of dot blot of each protein normalized to positive spot pixels.

Article Snippet: Profili cytokines expres ed by secretomes of irradiated or non-irradi ted fibro lasts obtained with Proteome Profile Human XL cytokine Array Kit (R&D) (A) Representative image of dot blot, (B) graph showing signal density of d t blot f r each protein normalized to positive spot pixels.

Techniques: Irradiation, Dot Blot

Journal: Biology

Article Title: Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium.

doi: 10.3390/biology11070980

Figure Lengend Snippet: Figure 9. MSC-EVs and MSC-CM protein composition. (A) Profiling of angiogenesis-regulating proteins secreted by MSC-EV or MSC-CM samples obtained with Proteome Profiler Human An- giogenesis Array Kit (R&D). At the top, representative image of dot blot and at the bottom, graph showing pixel density of dot blot of each protein normalized to positive spot pixels. (B) Profiling of cytokines secreted by MSC-EV or MSC-CM samples obtained with Proteome Profiler Human XL cytokine Array Kit (R&D). At the top, representative image of dot blot and at the bottom, graph showing pixel density of dot blot of each protein normalized to positive spot pixels (C): Quantification of IL8, MCP-1 and CXCL16 secreted by MSC-EV or MSC-CM samples by ELISA (mean ± SD), n = 3.

Article Snippet: Profili cytokines expres ed by secretomes of irradiated or non-irradi ted fibro lasts obtained with Proteome Profile Human XL cytokine Array Kit (R&D) (A) Representative image of dot blot, (B) graph showing signal density of d t blot f r each protein normalized to positive spot pixels.

Techniques: Dot Blot, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Macrophage migration inhibitory factor is overproduced through EGR1 in TET2 low resting monocytes

doi: 10.1038/s42003-022-03057-w

Figure Lengend Snippet: a , b Cytokine profile arrays of supernatant collected from cord blood CD34 + cells infected with SCR - and TET2- shRNA-GFP lentiviruses, sorted on GFP expression, and induced to differentiate with stem cell factor (SCF), interleukin-3 (IL-3), Fms-related tyrosine kinase 3 ligand (FLT3L) and granulocyte-colony stimulating factor (G-CSF). a Representative cytokine array with supernatants collected at day 10 of differentiation. The rectangle points to MIF detection. b Quantification of MIF signals, normalized to positive controls. Data are mean +/− SEM of three independent experiments. Paired t test: * P < 0.05. c MIF concentrations determined by ELISA in the supernatant of cells induced to differentiate for indicated time. Data are mean +/− SEM of indicated independent experiments (day 5: n = 6; day 7: n = 5; day 8: n = 3; day 10: n = 7). Paired t test: * P < 0.05; *** P < 0.001. d RT-qPCR analysis of MIF mRNA expression in four TET2 -depleted ( TET2 shRNA, gray bars) and control ( SCR shRNA, black bars) human leukemic cell lines. Data are mean +/− SEM of three biological replicates. Unpaired t test: * P < 0.05; *** P < 0.001; **** P < 0.0001. e Immunoblot of SCR or TET2 shRNA infected leukemic cell lines sorted on GFP expression. Lower panels, quantification after actin normalization using Image J software. f MIF concentrations determined by ELISA in the supernatants of kasumi-1 ( n = 5), M07e ( n = 5), UT-7 ( n = 4) and TF-1 ( n = 5) cells transduced 24 h before with SCR (black squares) or TET2 (gray squares) shRNA. Data are mean +/− SEM of indicated biological replicates. Unpaired-t test: * P < 0.05; ** P < 0.01. g MIF concentrations determined by ELISA in the plasma of two Tet2 -deficient models (1–3 months): K427 knock-out model (wt/wt or Tet2 +/+ n = 4, wt/exc or Tet2 +/− n = 5 and exc/exc or Tet2 −/− n = 5); ANO knock-down model (wt/wt or Tet2 + /+ n = 5, wt/LacZ or Tet2 +/− n = 5 and LacZ/LacZ or Tet2 −/− n = 6). Data are mean +/− SEM of indicated biological replicates. Dunnett’s multiple comparison tests using wt/wt as control: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CD34 + collected supernatants were analyzed using human cytokine antibody array (panel A; R&D Systems).

Techniques: Infection, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Western Blot, Software, Clinical Proteomics, Knock-Out, Knockdown, Comparison

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: GDF15 play a crucial role in the regulations of breast cancer cells activities by visfatin-treated ADSCs (vADSCs). ( A ) After the three-day co-culture of MDA-MB-231 cells and vADSCs (V50 and V100 group) or untreated ADSCs (Ctrl group), the CM was collected and analyzed by using a cytokine array kit. ( B ) The expression of GDF15 in the co-cultured CM was validated by ELISA represented in a histogram. The ADSCs and MDA-MB-231 cells collected from the co-culture system were extracted for cell lysate to analyze the GDF15 expression by western blotting. ( C ) The migration and invasion of MDA-MB-231 treated with GDF15 at various concentrations for 48 h were evaluated by using a transwell system. ( D ) The indirect co-culture was performed in the presence or absence of the GDF15 neutralizing antibody of for three days. After that, the migration and invasion of the MDA-MB-231 cells collected from the co-culture were evaluated in a transwell system. ( E ) The expression of phosphor-AKT (pAKT) of MDA-MB-231 treated with GDF15 (50 ng/mL) at different time point was detected by western blotting. ( F ) The pAKT was detected in the MDA-MB-231 cells from the co-culture by western blotting. ( G ) After the three-day co-culture in the presence or absence of the wortmannin (400 nM), the MDA-MB-231 cells were collected from the co-culture for performing the migration assay. All experiments were performed in triplicate.

Article Snippet: To explore the cytokine expression profile in the medium of the indirect co-culture of MDA-MB-231 and resistin-pretreated ADSCs, a Proteome profilerTM human XL cytokine array kit (ARY022, R&D Systems) was adopted.

Techniques: Co-Culture Assay, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Migration

Journal: Journal for immunotherapy of cancer

Article Title: Functional tumor cell-intrinsic STING, not host STING, drives local and systemic antitumor immunity and therapy efficacy following cryoablation.

doi: 10.1136/jitc-2022-006608

Figure Lengend Snippet: Figure 7 Preferential inflammatory chemokine and cytokine production in STINGwt RMS tumor microenvironment (TME) following cryoablation. C57BL/6 mice inoculated with 1×106 M3-9-MSTINGwt or M3-9-MSTING-/- were non-treated (non-cryo) or treated with cryoablation on day 10 when tumors reached 100 mm3. Five days later, tumors were harvested, weighed, minced, and suspended in PBS. (A) The supernatants were analyzed for IFN-β protein concentration by ELISA and normalized to tumor weight. For the chemokine and cytokine arrays, supernatants from each mouse were pooled by group, and the pooled supernatant for each group was then used to analyze chemokines (B) and cytokines (C, D). Densitometry was performed on the dot blot arrays (online supplemental figure S7) and normalized to average tumor weight, and results are displayed based on a relative intensity scale.

Article Snippet: 200 μL of pooled supernatant from each group was then used to perform the Proteome Profiler Mouse XL Cytokine Array (R&D Systems; Cat. No. ARY028) and 175 μL of pooled supernatant from each group was used to perform the Proteome Profiler Mouse Chemokine Array Kit (R&D Systems; Cat. No. ARY020), each according to manufacturer’s instructions.

Techniques: Protein Concentration, Enzyme-linked Immunosorbent Assay, Dot Blot

Journal: Science signaling

Article Title: Fusobacterium nucleatum host-cell binding and invasion induces IL-8 and CXCL1 secretion that drives colorectal cancer cell migration.

doi: 10.1126/scisignal.aba9157

Figure Lengend Snippet: Fig. 3. Cytokine secretion analysis from mouse neutrophils and macrophages. (A) Overview of experiments used to analyze Fnn-induced cytokine secretion from immune cells. (B) Heatmap of cytokine array assessing secretion of the indicated cytokines from mouse neutrophils in the presence of E. coli, Fnn, or deletion control (columns as designated by colored dots, legend right). Infection parameters were 50:1 MOI Fnn:neutrophils for 4 hours. (C) Fluorescence microscopy of Fnn interacting with mouse neutrophils. DNA was detected using DAPI, and Fnn was labeled with the fluorescent red membrane–intercalating dye FM 4-64FX. Images are represen- tative of n = 3 experiments. (D) ELISA confirming Fnn induction of CCL3 and CXCL2 and assessing the effect of deletion of fap2 on cytokine secretion in neutrophils. n = 3 independent experiments. (E) Cytokine array assessing the effect of Fnn infec- tion on the secretion of several cytokines from mouse macrophages when com- pared to that of E. coli and control. Sample conditions designated by colored dots as defined in (B); relative abundance scale as indicated, right. In (B) to (E), infection parameters were 50:1 MOI Fusobacterium:neutrophils/macrophages for 4 hours. nsNot significant (P > 0.05) by unpaired Student’s t test.

Article Snippet: The cytokine array membranes [R&D Systems: Proteome Profiler Human Cytokine Array (ARY005B), Proteome Profiler Mouse Cytokine Array Kit, Panel A (ARY006)] were then blocked for 1 hour in tris-buffered saline–3% bovine serum albumin (BSA) at room temperature on a rocking platform.

Techniques: Control, Infection, Fluorescence, Microscopy, Labeling, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Science signaling

Article Title: Fusobacterium nucleatum host-cell binding and invasion induces IL-8 and CXCL1 secretion that drives colorectal cancer cell migration.

doi: 10.1126/scisignal.aba9157

Figure Lengend Snippet: Fig. 2. F. nucleatum induces cytokine secretion from HCT116 cells. (A) Schematic of experiments used to analyze Fnn-induced cytokine secretion from HCT116 CRC cells. (B and C) Representative broad cytokine array dot blots [of 36 proteins; (B)] analyzing effect of infection with Fnn and Fnn-adhesin deletion strains. Control spots (B) indicate successful Western blots and provide densitometry controls for quantitation, calculated as fold increase and shown as a heatmap in (C). (D) IL-8 and CXCL1 ELISA to quantitate cytokine secretion from HCT116 cells induced by Fnn and Fnn ∆fap. n = 3 independent experiments. (E) IL-8 and CXCL1 ELISA to quanti tate and compare cytokine secretion from HCT116 cells induced by F. nucleatum subsp. nucleatum 25586, F. nucleatum subsp. nucleatum 23726, and F. nucleatum subsp. animalis 7_1 (Fna). n = 3 independent experiments. In (D) and (E), infec- tion parameters were 50:1 MOI Fusobacterium:HCT116 for 4 hours (tan) and 50:1 MOI Fusobacterium:HCT116 for 24 hours (purple). ****P < 0.0001 by unpaired Stu- dent’s t test or two-way ANOVA for single or grouped analyses, respectively.

Article Snippet: The cytokine array membranes [R&D Systems: Proteome Profiler Human Cytokine Array (ARY005B), Proteome Profiler Mouse Cytokine Array Kit, Panel A (ARY006)] were then blocked for 1 hour in tris-buffered saline–3% bovine serum albumin (BSA) at room temperature on a rocking platform.

Techniques: Infection, Control, Western Blot, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal for immunotherapy of cancer

Article Title: Ambra1 modulates the tumor immune microenvironment and response to PD-1 blockade in melanoma.

doi: 10.1136/jitc-2022-006389

Figure Lengend Snippet: Figure 4 Analysis of T cells migration upon Ambra1 deficiency. (A) Representative images of the Mouse Cytokine Array in supernatants (conditioned media) of Bdmc+/+ (left) and Bdmc−/− (right) cells. (B) The average pixel density (pair of duplicate spots represents each cytokine) was quantified and reported as fold change compared with Bdmc+/+. The heat map of the less secreted cytokines by Bdmc−/− cells compared with Bdmc+/+ is shown. (C) Gene set analysis (GSA) shows the downregulated pathways in Ambra1 deficiency conditions (p<0.01) based on the differentially expressed cytokines in (B). Out of 33 cytokines included in the panel, 28 were found in the pathway database. For GSA, hypergeometric tests were used and genes with a p value<0.01 and abs(log FC)>1 were considered as significant (BioCarta and WikiPathways). (D) Splenocytes were cultured in Bdmc+/+, Bdmc−/− or Bdmc−/−/Il-1α- (10 ng/mL) derived conditioned media for 26–28 hours and migration of CD3+CD8+, CD3+CD4+ and CD3+CD4+CD25+ T cells examined by flow cytometry. Error bars indicate SEM (*p<0.05, ***p<0.001, n=6–9). (E) RT-qPCR analysis of Ccl22 in splenocytes (n=3) after Il-1α administration at indicated times. Data were normalized to the internal control L34 and expressed as fold change±SEM with respect to the untreated splenocytes (ctr) (*p<0.05, 3 hours or 24 hours vs ctr). (F) AMBRA1 was silenced in SK-Mel-5 (siAMBRA1) for 48 hours and WB analyses (n=3) performed to evaluate silencing (TE, total extracts). Actin was used as loading control. Data in the graph represent densitometry analyses and are shown as fold change±SD versus siCT cells (**p<0.01). (G) Representative WB analyses (n=3) of total (TE), cytosolic (CE) and nuclear (NE) extracts of SK-Mel-5 silenced for AMBRA1 as shown in (F). FAK1 was detected together with the purity controls lamin A/C (for TE) and LDH (for CE). (H) The cytosolic and nuclear fractions of FAK1 shown in (G) were quantified. Data are expressed as average±SD (ns, not significant). (I) Representative WB analyses (n=3) of SK-Mel-5 re-expressing myc-tagged AMBRA1 mutants (WT and L110F). AMBRA1 and c-myc were detected to determine transfection efficiency; LC3 as marker of the autophagy flux; actin as loading control. (J) The autophagy marker LC3 shown in (I) was quantified. Data are expressed as fold change±SD (n=3; *p<0.05). (K) Secretion of IL-1α was measured in supernatants of cells transfected as in (I). Each dot represents the average pixel density of a single membrane’s duplet. Data are expressed as arbitrary unit ±SD (n=2; ns, not significant). (L) RT-qPCR analysis of IL-1α in SK-Mel-5 transfected as in (I). Data were normalized to the internal control RPLP0 and expressed as fold change±SEM (**p<0.01). Ambra1, autophagy and beclin 1 regulator 1; Bdmc, BPA-derived melanoma cells; FAK1, focal adhesion kinase 1; IL, interleukin; LDH, lactate dehydrogenase ; RT-qPCR, real-time quantitative PCR ; WB, western blot.

Article Snippet: The profiling of different cytokines and chemokines was performed using the Proteome Profiler Mouse/Human Cytokine Array Kit (R&D systems, cat# ARY006 and cat# ARY005B, respectively) according to the manufacturer’s instructions.

Techniques: Migration, Cell Culture, Derivative Assay, Flow Cytometry, Quantitative RT-PCR, Control, Expressing, Transfection, Marker, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell Reports

Article Title: Human Induced Pluripotent Stem Cell-Derived Microglia-Like Cells Harboring TREM2 Missense Mutations Show Specific Deficits in Phagocytosis

doi: 10.1016/j.celrep.2018.07.094

Figure Lengend Snippet: Analysis of Secreted Factors from Controls and TREM2 Variant iPSC-MGLCs after LPS Exposure (A) Proteome profiles of secreted factors from control, T66Mhet, T66Mhom, and W50Chom iPSC-MGLCs analyzed using a Human XL Cytokine Array Kit (R&D Systems). Representative dot blots from iPSC-MGLC culture supernatants from one control line, one T66Mhet line, one T66Mhom, and W50Chom iPSC-MGLCs are shown under basal (untreated) conditions and LPS-treated conditions; 100 ng/mL after 18 hr. (B and C) Analysis of dot blot intensity from supernatants of basal (B) and LPS-treated (C) cultures. Mixed supernatants from independent experiments were used; n = 3 for all genotypes. (D) Levels of 10 cytokines, including TNF-α, IL-6, CHI3L1, IL-1β, IL-4, and IL-12p70 were measured simultaneously on the Meso Scale Diagnostics (MSD) platform using a V-PLEX Pro-inflammatory Panel 1 (n = 2 for untreated, n = 3 for LPS-treated samples in four control iPSC lines, and two clones per TREM2 variant iPSC line were analyzed; data are presented as mean ±SEM). See also Figure S6 .

Article Snippet: Proteome Profiler Human XL Cytokine antibody array membranes or human cytokine array membranes (R&D Systems) were incubated with supernatants pooled from three independent experiments, according to TREM2 genotype after basal, LPS, or apoptotic cell treatment, as per the manufacturer’s instructions.

Techniques: Variant Assay, Control, Dot Blot, Clone Assay

Journal: Cell Reports

Article Title: Human Induced Pluripotent Stem Cell-Derived Microglia-Like Cells Harboring TREM2 Missense Mutations Show Specific Deficits in Phagocytosis

doi: 10.1016/j.celrep.2018.07.094

Figure Lengend Snippet: Apoptotic Cell-Mediated Cytokine Responses Are Dependent on Genotype (A) Proteome profiles of secreted factors from control, T66Mhet, and T66Mhom iPSC-MGLCs were analyzed using a human cytokine array. Representative dot blots from pooled cell culture supernatants from two control, T66Mhet, and T66Mhom lines, and apoptotic cells only are shown under basal (untreated) conditions and following exposure to apoptotic SH-SY5Y cells for 48 hr. (B) Analysis of dot blot intensity from supernatants of basal cell- and apoptotic cell-exposed cultures revealed modulation of secreted factors associated with chemotaxis and chemoattraction with a dependence on genotype (C and D) Mixed supernatants from independent experiments were used; n = 3 for all genotypes. Representative images from cell culture scratch assays. Scale bar: 200 μm (C). Cultures were imaged live after 24h exposure to trans-wells containing apoptotic SH-SY5Y and subsequent Hoechst staining (D). Analysis was performed using ImageJ thresholding and particle quantification of the scratch area in (D). Two-way ANOVA with Bonferroni multiple comparison was used for analysis. Data are presented as means ± SEMs from two control lines, and one T66Mhet, and one T66Mhom line; n = 3 ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). See also Figure S7 .

Article Snippet: Proteome Profiler Human XL Cytokine antibody array membranes or human cytokine array membranes (R&D Systems) were incubated with supernatants pooled from three independent experiments, according to TREM2 genotype after basal, LPS, or apoptotic cell treatment, as per the manufacturer’s instructions.

Techniques: Control, Cell Culture, Dot Blot, Chemotaxis Assay, Staining, Comparison