Journal: bioRxiv

Article Title: Metastasis-initiating osteosarcoma subpopulations establish paracrine interactions with both lung and tumor cells to create a metastatic niche

doi: 10.1101/2024.06.09.597967

Figure Lengend Snippet: IL1α-induced IL6 and CXCL8 production requires NFkB signaling in a subpopulation of cells in G1 cell cycle phase. A) OS-17 cells were stimulation with IL1α, then fixed and stained for NFkB (green) or IL1R1 (red) at indicated timepoints. Note nuclear translocation of NFkB. Scale bar= 50µm. B) IKK inhibitors IKK-16 and TPCA-1 abrogate IL1α-induced IL6 and CXCL8 secretion as measured by ELISA. n=2 biological replicates done in technical triplicates. C) Single-cell RNA sequencing analysis of OS-17 tibial xenograft tumors, OS-17 experimental lung metastasis, human primary tumors, and human lung metastasis samples demonstrating restriction of NFkB activity (see methods) in small subpopulation of G1 cells.

Article Snippet: Following incubation, cell-free supernatants were collected and analyzed by ELISA for changes in concentration of IL6 and CXCL8 using DuoSet ELISA Development Kits (R&D Systems, DY206, DY208).

Techniques: Staining, Translocation Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Metastasis-initiating osteosarcoma subpopulations establish paracrine interactions with both lung and tumor cells to create a metastatic niche

doi: 10.1101/2024.06.09.597967

Figure Lengend Snippet: Sustained IL6 production is maintained by a small subset of hypo-proliferative cells that anchor osteosarcoma cells to the lung niche. A) Immunofluorescence expression of IL6(red) and CXCL8 (green) over time (8-72 hours) following stimulation with IL1α. White marks proliferating cells (Ki67). Blue=DAPI. B-C) ELISA and immunofluorescence quantitation demonstrating that IL6 and CXCL8 production remains constant over time while IL6 and CXCL8+ expression by immunofluorescence becomes progressively restricted to Ki67-cells. ELISA= 2 biological replicates done in technical triplicate. Percentage of IL6, CXCL8, and Ki67 quantified in n=3 experiments, 100 cells/experiment. D) IL6, CXCL8, and Ki67 expression in NCH OS-4 cells following stimulation with IL1α (72 hours). E) F420 murine cells stimulated with murine IL1α (72 hours) and stained with Ki67 (red), IL6 (green), and DAPI (blue). Scale bar= 50µm. F) quantitation of IL6 and Ki67 staining in NCH OS-4 and F420 cells.

Article Snippet: Following incubation, cell-free supernatants were collected and analyzed by ELISA for changes in concentration of IL6 and CXCL8 using DuoSet ELISA Development Kits (R&D Systems, DY206, DY208).

Techniques: Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining

Journal: bioRxiv

Article Title: Metastasis-initiating osteosarcoma subpopulations establish paracrine interactions with both lung and tumor cells to create a metastatic niche

doi: 10.1101/2024.06.09.597967

Figure Lengend Snippet: IL1 signaling is required for osteosarcoma metastasis progression. A) Functional validation of IL1R1 CRISPR knock-out by loss of epithelial-induced IL6 secretion measured by ELISA. n=4 biological replicates done in triplicate. B) Representative H/E stained lungs from mice inoculated OS-17 electroporated control (NG) or OS-17 IL1R1 CRISPR knock-out cell lines B and C. C) Number of metastatic lesions/ slice quantified by blinded examiner. n=15 mice/condition. **** p<0.0001. Welch’s t-test. D-E) Representative images and quantification of number of metastatic lesions and percent metastasis burden (relative to whole lung area) in mice inoculated via tail vein with OS-17 cells then treated with vehicle (PBS) or anakinra 1 day following tumor injection. **p<0.01. Welch’s t-test. F-G) Representative images and quantification of number of metastatic lesions and percent metastasis burden (relative to whole lung area) in mice inoculated via tail vein with F420 cells then treated with vehicle (PBS) or anakinra 1 day following tumor injection. *p<0.05. Welch’s t-test. Scale bar= 2mm.

Article Snippet: Following incubation, cell-free supernatants were collected and analyzed by ELISA for changes in concentration of IL6 and CXCL8 using DuoSet ELISA Development Kits (R&D Systems, DY206, DY208).

Techniques: Functional Assay, Biomarker Discovery, CRISPR, Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Control, Injection

Journal: The Journal of Clinical Investigation

Article Title: TET3-overexpressing macrophages promote endometriosis

doi: 10.1172/JCI181839

Figure Lengend Snippet: ( A ) qRT-PCR of Il1b and Il6 mRNAs of cultured PMs isolated from Mye-Tet3–KO mice or WT controls and treated with 10 ng/mL LPS plus 20 ng/mL IFN-γ. RNAs were isolated after 6 hours of LPS/IFN-γ stimulation. Uns, unstimulated. n = 3 mice per genotype. ( B ) ELISA analysis (after 6 hours of LPS/IFN-γ stimulation) of IL-1β and IL-6 of cultured PMs treated as in A . n = 3 mice per genotype. ( C ) Human MDMs primed with 10 ng/mL of TGF-β1 were transfected with NT siRNA or TET3 siRNA. After 48 hours of transfection, cells were stimulated with 10 ng/mL LPS plus 20 ng/mL IFN-γ for 8 hours, followed by RNA extraction and qRT-PCR of IL-1B and IL-6 mRNAs. n = 3 biological replicates. ( D ) ELISA analysis (after 8 hours of LPS/IFN-γ stimulation) of IL-1β and IL-6 of MDMs following treatment as in C . ( E ) Human MDMs were infected with Ad-GFP or Ad-TET3. The next day, proteins were extracted, followed by Western blot analysis. The Ad-TET3–infected cells showed approximately 5-fold TET3 overexpression as compared with Ad-GFP–infected cells. ( F ) Human MDMs were infected with Ad-GFP or Ad-TET3. The next day, cells were stimulated with 10 ng/mL LPS plus 20 ng/mL IFN-γ for 8 hours, followed by RNA extraction and qRT-PCR of IL-1B and IL-6 mRNAs. n = 3 biological replicates. ( G ) ELISA analysis (after 8 hours of LPS/IFN-γ stimulation) of IL-1β and IL-6 of MDMs following treatment as in F . All data are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, 2-tailed Student’s t test.

Article Snippet: IL-1β and IL-6 protein levels in the supernatant of cultured mouse PMs were measured using ELISA kits (R&D Systems, MLB00C and M6000B-1).

Techniques: Quantitative RT-PCR, Cell Culture, Isolation, Enzyme-linked Immunosorbent Assay, Transfection, RNA Extraction, Infection, Western Blot, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: TET3-overexpressing macrophages promote endometriosis

doi: 10.1172/JCI181839

Figure Lengend Snippet: ( A ) qRT-PCR of let-7a in RAW 264.7 cells transfected with NT siRNA or Tet3 siRNA. RNA was isolated at 24 hours after transfection. n = 3 technical replicates. ( B ) qRT-PCR of let-7a in PMs isolated from WT and Mye-Tet3–KO mice. n = 3 mice per genotype. ( C ) PMs were isolated from WT mice and treated with TGF-β1 at a final concentration of 30 ng/mL. After 48 hours, Veh or Bc was added at a final concentration of 10 μM and incubation carried out for 48 hours. RNAs were extracted and analyzed by qRT-PCR. n = 3 mice per group. ( D ) qRT-PCR of Lin28b mRNA in PMs isolated from WT and Mye-Tet3–KO mice. n = 3 mice per genotype. ( E ) qRT-PCR of Lin28b mRNA in PMs isolated from WT mice and treated as in C . n = 3 mice per group. ( F ) qRT-PCR of Il-1b and Il-6 mRNAs of PMs isolated from WT mice and transfected with control miRNA (miCon) or let-7a mimic and stimulated with 10 ng/mL LPS plus 20 ng/mL IFN-γ. RNAs were isolated after 6 hours of LPS/IFN-γ stimulation. n = 3 mice per group. ( G ) ELISA results of IL-1β (after 6 hours of LPS/IFN-γ stimulation) and IL-6 (after 10 hours of LPS/IFN-γ stimulation) of PMs treated as in F . n = 3 mice per group. ( H ) Relative let-7a miRNA levels in endometriosis lesions from WT and Mye-tet3–KO mice. n = 4 animals per genotype. ( I ) Relative let-7a miRNA levels in endometriosis lesions from Veh- or Bc-treated mice. n = 4 animals per group. All data are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, 2-tailed Student’s t test.

Article Snippet: IL-1β and IL-6 protein levels in the supernatant of cultured mouse PMs were measured using ELISA kits (R&D Systems, MLB00C and M6000B-1).

Techniques: Quantitative RT-PCR, Transfection, Isolation, Concentration Assay, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: 3D co-cultures of primary human hepatocytes and Kupffer-like cells to address innate immune responses to rAAV

doi: 10.1038/s41598-025-31044-8

Figure Lengend Snippet: 3D PHH and 3D PHH:KLC cultures are responsive to PAMPs. Gene expression evaluation of PRRs and cytokines after stimulation with prototypical stimuli for 6 h in PHH and PHH:KLC (13 days of 3D culture). Cultures were stimulated with: ( a ) LPS 1 µg/mL, ( b ) Pam3CSK4 1 µg/mL. Data are expressed as relative mRNA abundance relative to 36B4; N ≥ 3. Paired T-test was performed for statistical analysis. ( c ) Evaluation of IL-6 secretion in PHH and PHH:KLC 3D cultures after stimulation with LPS 1 µg/mL or Pam3CSK4 1 µg/mL; N = 3. One way ANOVA was employed for statistical analysis. In all graphs, different symbols correspond to different PHH:KLC donor combinations.

Article Snippet: The concentration of the inflammatory mediator IL-6 in the supernatant of 3D cultures stimulated with 1 μg/mL LPS, 1 μg/mL Pam3CSK4 and rAAV (MOI 1 × 10 6 VG/cell) were measured by using the Human IL-6 ELISA Kit (R&D Systems) following manufacturer’s instructions.

Techniques: Gene Expression

Journal: Scientific Reports

Article Title: 3D co-cultures of primary human hepatocytes and Kupffer-like cells to address innate immune responses to rAAV

doi: 10.1038/s41598-025-31044-8

Figure Lengend Snippet: 3D PHH:KLC co-cultures are susceptible and responsive to rAAV8. ( a ) Evaluation of mCherry gene expression after PHH:KLC 3D culture transduction with rAAV8-mCherry for 6 and 24 h. Results are expressed as fold change in respect to the vehicle condition; N = 3. ( b ) Immunofluorescence confocal microscopy evaluation of PHH:KLC 3D co-cultures transduced with rAAV8-mCherry (MOI 1 × 10 6 VG/cell). Myeloid cells were intracellularly labelled with Cell Tracker TM Deep red (green) and nuclei were stained with DAPI (blue). Transgene expression was assessed by mCherry detection (red); 1 stack image, scale bar = 50 µm. ( c ) Gene expression evaluation of cytokines after stimulation with rAAV8-mCherry for 6 h in PHH and PHH:KLC (13 days of 3D culture). Results are normalized for a housekeeping gene expression (36B4) and expressed as fold change in respect to the vehicle condition; N ≥ 8. Unpaired T-test was performed for statistical analysis. ( d ) Gene expression evaluation of cytokines after stimulation with rAAV8-mCherry for 6 and 24 h in PHH:KLC (13 days of 3D culture). Results are normalized for a housekeeping gene expression (36B4) and expressed as fold change in respect to the vehicle condition; N ≥ 6. Paired T-test was performed for statistical analysis. A total of 7 PBMC donors combined with 4 PHH donors were employed. ( e ) IL-6 secretion evaluation after PHH:KLC 3D culture challenge with rAAV8-mCherry for 6 and 24 h. Paired T-test was performed for statistical analysis; N ≥ 3. In all graphs, symbols correspond to different PHH:KLC donor combinations. For box plots, the whiskers denote minimum and maximum values and the horizontal line the median.

Article Snippet: The concentration of the inflammatory mediator IL-6 in the supernatant of 3D cultures stimulated with 1 μg/mL LPS, 1 μg/mL Pam3CSK4 and rAAV (MOI 1 × 10 6 VG/cell) were measured by using the Human IL-6 ELISA Kit (R&D Systems) following manufacturer’s instructions.

Techniques: Gene Expression, Transduction, Immunofluorescence, Confocal Microscopy, Staining, Expressing

Journal: Journal of neuroinflammation

Article Title: Complex molecular and functional outcomes of single versus sequential cytokine stimulation of rat microglia.

doi: 10.1186/s12974-016-0531-9

Figure Lengend Snippet: Fig. 3 Expression of ROS-related molecules and contribution of NOX enzymes to myelin phagocytosis and ROS production. a Expression of ROS-related molecules. Rat microglia were stimulated with cytokines for 24 h with or without a subsequent 6-h exposure to myelin (plus or minus sign indicates presence/absence of myelin), as in Figs. 1a and 2a. b Effect of NOX inhibition on phagocytosis of myelin fragments. The activation treatments, assay, and normalization of data were the same as Fig. 2b, but now comparing the pan-NOX inhibitor, 5 μM VAS2870. c Effect of NOX inhibition on ROS levels in microglia that were treated as in Fig. 2b. As above, ROS levels were normalized to unstimulated microglia without myelin (dashed line), data are expressed as mean ± SEM (n = 6 individual cultures), and results were analyzed by two-way ANOVA with Bonferroni’s post hoc test. The comparisons are as follows: asterisk unstimulated (CTL) versus different activation states in the absence of myelin, dagger sign unstimulated (CTL) versus different activation states in the presence of myelin, number sign effects of myelin (a) or VAS2870 (b, c) within a particular activation state. One symbol p < 0.05, two symbols p < 0.01, three symbols p < 0.001

Article Snippet: To induce different activation states, microglia were exposed to recombinant rat cytokines (R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Inhibition, Activation Assay

Journal: Journal of neuroinflammation

Article Title: Complex molecular and functional outcomes of single versus sequential cytokine stimulation of rat microglia.

doi: 10.1186/s12974-016-0531-9

Figure Lengend Snippet: Fig. 8 Transcript expression of K+ channels and SOCE-related genes is affected by the microglial activation state and myelin phagocytosis. a Single cytokines with or without myelin. Microglia were stimulated for 24 h with 20 ng/mL IFN-γ + 50 ng/mL TNF-α (I + T) or 20 ng/mL IL-4, and then treated with 25 μg/mL myelin for 6 h. b M2a→M1 paradigm. Microglia were treated with 20 ng/mL IL-4 for 2 h followed by I + T for 22 h. c M1→M2 paradigm. Cells were treated with I + T for 2 h followed by IL-4 (M1→M2a) or 20 ng/mL IL-10 (M1→M2c) for 22 h. All data are expressed as mRNA counts in 200 ng total RNA (mean ± SEM) for six individual cultures. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. The dashed lines in b and c indicate expression levels in unstimulated (control) microglia. Comparisons are as follows: asterisk differences from control microglia, dagger sign CTL versus different activation states in the presence of myelin, number sign effects of myelin within a particular activation state (a) or effects of a second stimulus on the first stimulus (b and c). One symbol p < 0.05, two symbols p < 0.01, three symbols p < 0.001

Article Snippet: To induce different activation states, microglia were exposed to recombinant rat cytokines (R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Activation Assay, Control