Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: CRISPR-mediated MECOM depletion retards tumor growth by reducing cancer stem cell properties in lung squamous cell carcinoma.

doi: 10.1016/j.ymthe.2022.06.011

Figure Lengend Snippet: Figure 5. MECOM depletion suppressed CSC-associated factors (A and B) ADV-CRISPR-SaCas9 with MECOM depletion (MECOM-KO) was intratumorally injected into the PDX models. After 2 days, the tumors were isolated from the LUSC006, LUSC018, and LUSC021 cases for gene editing analyses using T7EI assay (A: LUSC006, LUSC018, and LUSC021) and Sanger sequence (B: LUSC021). (C–E) After 24–28 days, the treated tumors were isolated from the LUSC006, LUSC018, and LUSC021 cases for western blot and immunohistochemistry assays. Western blots (C) show MECOM, SOX2, ABCG2, CD44, and b-actin expression in the tumor samples of LUSC006, LUSC018, and LUSC021 models with depleted MECOM (ME- COM-KO). b-Actin was used as the loading control. The numerical value under the band shows densitometric analyses of the indicated protein expression, compared to the corresponding control, which was normalized as “1.0.” Photographs (D) and IRS scores (E) of MECOM, CD44, and ABCG2 in the tumor samples of LUSC006, LUSC018, and LUSC021 models with MECOM-depleted ADV (MECOM-KO) by immunohistochemistry analyses. Scale bars, 50 mm. *p ＜0.05, unpaired 2-tailed t test.

Article Snippet: Briefly, the slides were blocked with normal goat serum and stained with the primary antibodies of MECOM (28100002, Novus Biologicals), CD44 (15675-1-AP, Proteintech), and ABCG2 (27286-1-AP, Proteintech).

Techniques: CRISPR, Injection, Isolation, T7EI Assay, Sequencing, Western Blot, Immunohistochemistry, Expressing, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: CRISPR-mediated MECOM depletion retards tumor growth by reducing cancer stem cell properties in lung squamous cell carcinoma.

doi: 10.1016/j.ymthe.2022.06.011

Figure Lengend Snippet: Figure 7. Intravenous administration of MECOM depletion by ADV, adaptor, and protector complex (A and B) A diagram (A) for therapeutic treatment with the complex CFS, HF, and ADV with depleted MECOM in NOD-SCID mice (LUSC021 PDX model) via tail-vein injection. MECOM-depleted ADV was incubated with CFS and HF to form ADV/protein complex for 2 h. The complex was injected into the NOD-SCID mice via tail vein for 4 rounds with a 12-day interval. The ADV amount was 0.7 1010 VP, and the protein concentration was 1.0 107 pmol/VP. The growth curve (B) of LUSC021 PDX mice with the ADV/ protein complex (MECOM-KO) via tail-vein injection. The ADV/protein complex expressing non-targeting sgRNA was used as the control. n = 4, *p ＜0.05, unpaired 2-tailed t test. (C) The image of T7EI detection in the ADV/protein-treated tumors after administration for 48 h. (D) Immunohistochemistry staining shows MECOM, ABCG2, and CD44 expression in the tumors with treatment of ADV/protein. Scale bar, 50 mm. (E) Western blots demonstrate the indicated protein expression in the tumors with treatment of ADV/protein. b-Actin was used as loading control. The numerical value under the band shows densitometric analyses of the indicated protein expression, compared to the corresponding control, which was normalized as “1.0.” (F–H) The ADV viruses including control, MECOM depletion (MECOM-KO), and MECOM-KO plus SOX2 overexpression (MECOM-KO + SOX2) were intratumorally in- jected into the NOS-SCID mice (LUSC021). The ADV was intratumorally injected at days 0 and 12 with the amounts of 1.0 1010 VP and 0.5 1010 VP, respectively. Tumor image (F), growth curve (G), and tumor weight graph (H) of LUSC021 PDX model with indicated treatment. Scale bar, 1 cm. n = 5, *p ＜0.05, unpaired 2-tailed t test.

Article Snippet: Briefly, the slides were blocked with normal goat serum and stained with the primary antibodies of MECOM (28100002, Novus Biologicals), CD44 (15675-1-AP, Proteintech), and ABCG2 (27286-1-AP, Proteintech).

Techniques: Injection, Incubation, Protein Concentration, Expressing, Control, Immunohistochemistry, Staining, Western Blot, Over Expression

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: CRISPR-mediated MECOM depletion retards tumor growth by reducing cancer stem cell properties in lung squamous cell carcinoma.

doi: 10.1016/j.ymthe.2022.06.011

Figure Lengend Snippet: Figure 8. Intravenous administration of MECOM-depleted ADV/protein complex in orthotopic xenograft model (A–C) A diagram (A) for intravenous administration of ADV/protein complex with MECOM depletion in H520 orthotopic xenograft (NOD-SCID mice). MECOM-depleted ADV was incubated with CFS and HF proteins to form ADV/protein complex for 2 h. The complex was injected into the mice via tail vein for 2 rounds with a 14-day interval. The ADV amount was 0.7 1010 VP, and the protein concentration was 1.0 107 pmol/VP. The bioluminescent imaging (BI) was taken on days 0, 14, and 28 after ADV delivery to demonstrate the tumor growth. Bioluminescent images (B) and growth curves (C) of orthotopic tumors treated with MECOM-depleted ADV/protein complex (MECOM-KO) via tail-vein injection. n = 4. *p ＜0.05, unpaired 2-tailed t test. (D) Immunohistochemistry staining demonstrates MECOM, ABCG2, and CD44 expression in the H520 orthotopic xenografts treated with ADV/protein complex including control and MECOM depletion groups. Scale bar, 100 mm.

Article Snippet: Briefly, the slides were blocked with normal goat serum and stained with the primary antibodies of MECOM (28100002, Novus Biologicals), CD44 (15675-1-AP, Proteintech), and ABCG2 (27286-1-AP, Proteintech).

Techniques: Incubation, Injection, Protein Concentration, Imaging, Immunohistochemistry, Staining, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: Pigment Epithelium-derived Factor (PEDF) Promotes Tumor Cell Death by Inducing Macrophage Membrane Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)

doi: 10.1074/jbc.m111.266064

Figure Lengend Snippet: FIGURE2.PEDFinducesexpressionsofTRAILandPPARinBMDMs.A,BMDMsweretreatedwithPEDFattheindicatedconcentrationsfor24h,andthecells were then processed for RT-PCR analysis. GAPDH expression was examined for normalization purposes. B, cells were treated as described above, and proteins were detected by Western blot analysis with antibodies as indicated. Representative blots (left panels) and densitometric analysis with S.D. (error bars) (right panels) of three independent experiments are shown. *, p 0.05 versus untreated cells. C, BMDMs were exposed to PEDF for the time indicated and then harvested for Western blotting with antibodies against TRAIL (33.4 kDa) and PPAR (57.6 kDa). Equal protein loading was confirmed by reprobing the membranes with a -actin antibody. Representative blots and densitometric analyses with S.D. from four separate experiments are shown. D, PEDF-treated BMDMs were assessed for cell surface TRAIL expression. BMDMs were treated with PEDF or its solvent for 24 h. Cell membrane and cytosolic fractions were isolated as described under “Experimental Procedures” and then subjected to Western blot analysis. A representative result from two independent experi- ments is shown. E, BMDMs were treated with PEDF or solvent control for 24 h. The cells were then incubated with PE-conjugated isotypic control or PE- conjugated TRAIL antibody and analyzed by flow cytometry.

Article Snippet: Transfection of Short Interfering RNA (siRNA)—Subconfluent BMDMs were transfected with a mixture of mouse PPAR siRNAs (sc-29456; Santa Cruz Biotechnology, Inc.) using Lipofectamine RNAiMAX reagent (Invitrogen) in serum-free medium according to the manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Solvent, Membrane, Isolation, Control, Incubation, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Pigment Epithelium-derived Factor (PEDF) Promotes Tumor Cell Death by Inducing Macrophage Membrane Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)

doi: 10.1074/jbc.m111.266064

Figure Lengend Snippet: FIGURE 3. PEDF mediates the induction of TRAIL expression by PPAR. A, PPAR antagonists suppress PEDF-induced TRAIL expression. BMDMs were pretreated with 10 M GW9662 or G3335 for 1 h and then treated with or without 200 ng/ml PEDF (P) for an additional 24 h. Cells were harvested for Western blot analysis. Loading equality was confirmed with antibodies against -actin. Representative blots and densitometric analyses with S.D. (error bars) from three separate experiments are shown. #, p 0.005 versus PEDF DMSO. B, surface expression of TRAIL was quantified by flow cytometry. BMDMs were exposed to PEDF or PEDF solvent for 24 h or pretreated with 10 M GW9662 for 1 h before exposure to PEDF for an additional 24 h. The cells were then stained with PE-conjugated isotypic control or anti-TRAIL antibody for analysis by flow cytometry. Data shown are from one representative experiment of four. C, PPAR siRNA abrogates PEDF-induced TRAIL expression. BMDMs were transfected with a PPAR siRNA or control siRNA for 16 h and allowed to recover for a further 24 h. Mock, cells were treated with transfection reagents alone. After the respective treatment, both BMDMs and siRNA-transfected BMDMs were exposed to PEDF for 24 h, and the cells were then harvested for Western blot analysis (blots 1–3). The siRNA-transfected BMDMs were also used for coculture with TC-1 cells at an effector/target ratio of 25:1 for 16 h and then exposed to PEDF for a further 24 h, followed by detection of activated caspase-3 by Western blot analysis. Representative results from three separate experiments are shown. D, PPAR antagonist and siRNA block the BMDM-mediated cytotoxicity induced by PEDF, GW9662, and siRNAs pretreatments were performed as described above, followed by PEDF treatment for an additional 24 h. BMDM-mediated cytotoxicity was performedataneffector/targetratioof25:1,andcellapoptosiswasdetectedbyannexinV-FITCstainingasdescribedinthelegendtoFig.1A.*,p 0.001versus PEDF DMSO. **, p 0.02 versus control siRNA PEDF.

Article Snippet: Transfection of Short Interfering RNA (siRNA)—Subconfluent BMDMs were transfected with a mixture of mouse PPAR siRNAs (sc-29456; Santa Cruz Biotechnology, Inc.) using Lipofectamine RNAiMAX reagent (Invitrogen) in serum-free medium according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Flow Cytometry, Solvent, Staining, Control, Transfection, Blocking Assay

Journal: Journal of Biological Chemistry

Article Title: Pigment Epithelium-derived Factor (PEDF) Promotes Tumor Cell Death by Inducing Macrophage Membrane Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)

doi: 10.1074/jbc.m111.266064

Figure Lengend Snippet: FIGURE6.A,sequenceoftheconsensusPPREandsequenceofthecandidatePPREcontainedwithinthehumanTRAILpromoter5-flankingregion.Thelocation of the candidate PPRE (nt 382/370) is marked with an arrow. The candidate PPRE mutated from GT to CA is designated as PPRE-m. Numbers are relative to the transcription start site (1). The region spanning 1594 bp of the promoter was progressively deleted from its 5-end and fused to the pGL3 basic vector. B, PPAR transactivates the human TRAIL promoter. HuH-7 cells were transiently transfected with the indicated reporter construct in the presence or absence of the PPAR-expressing plasmid, pcDNA-P. Black columns represent cells that were treated with 20 M GW9662 during transfection. Values (mean S.D. (error bars)) represent firefly luciferase activity normalized relative to a Renilla luciferase internal control. Luciferase activities are shown relative to the activity of the TRAILp vector, which was arbitrarily set to 1. *, p 0.05 versus. TRAILp. #, p 0.05 versus pcDNA-P TRAILp. ##, p 0.001 versus pcDNA-P TRAILp.

Article Snippet: Transfection of Short Interfering RNA (siRNA)—Subconfluent BMDMs were transfected with a mixture of mouse PPAR siRNAs (sc-29456; Santa Cruz Biotechnology, Inc.) using Lipofectamine RNAiMAX reagent (Invitrogen) in serum-free medium according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Transfection, Construct, Expressing, Luciferase, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Pigment Epithelium-derived Factor (PEDF) Promotes Tumor Cell Death by Inducing Macrophage Membrane Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)

doi: 10.1074/jbc.m111.266064

Figure Lengend Snippet: FIGURE 7. PPAR binds to the TRAIL promoter. A, PEDF enhances human TRAIL promoter activity. THP-1 macrophages were transfected with the indicated reporter construct. After 48 h post-transfection, cells were stimulated with PEDF or PEDF combined with 10 M GW9662 for a further 24 h prior to reporter gene activity assays as described above. *, p 0.05 versus. TRAILp. #, p 0.05 versus PEDF TRAILp. ##, p 0.05 versus PEDF TRAILp. B, ChIP assay. THP-1 macrophages were treated with PEDF (P) or PEDF solvent (S) for 24 h or pretreated with 10 M GW9662 for 1 h and then incubated with PEDF for an additional 24 h. PPAR-DNA complexes were immunoprecipitated with the anti-PPAR antibody or IgG. Data are normalized to IgG immunoprecipitated DNA and input DNA. The binding of PPAR to the TRAIL promoter in PEDF-stimulated THP-1 macrophages was measured and quantified by real-time PCR. C and D, identifi- cation of PPAR and PPRE association by DNA pull-down assay. The 5-biotinylated double-stranded oligonucleotides containing a consensus PPRE (positive control(PC))orTRAILpPPREoritsmutant,asindicatedinFig.6A,wereusedtoprecipitatePPARandRXRfromnuclearproteinextractsofTHP-1macrophages treated with PEDF or its solvent. The mixtures were mixed with or without PPAR antagonists (reaching a final concentration of 20 M) and then pulled down by streptavidin beads. The proteins in the complex were analyzed by Western blotting using antibodies against PPAR or RXR isoforms. Aliquots of nuclear extracts were also analyzed before the DNA pull-down assay (input). Shown are representative experiments that were repeated four times with similar observations. Error bars, S.E.

Article Snippet: Transfection of Short Interfering RNA (siRNA)—Subconfluent BMDMs were transfected with a mixture of mouse PPAR siRNAs (sc-29456; Santa Cruz Biotechnology, Inc.) using Lipofectamine RNAiMAX reagent (Invitrogen) in serum-free medium according to the manufacturer’s instructions.

Techniques: Activity Assay, Transfection, Construct, Solvent, Incubation, Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Pull Down Assay, Positive Control, Concentration Assay, Western Blot

Journal: Frontiers in Physiology

Article Title: Conduction defects and arrhythmias in mdx mice are not associated with a degeneration of the cardiac Purkinje network

doi: 10.3389/fphys.2025.1607916

Figure Lengend Snippet: No morphological and maturation defects of the Purkinje Fiber network enriched in dystrophin. (a) Whole-mount immunofluorescence with Cx43 antibodies and Cx40-GFP on opened LV from WT and mdx adult mice. On the left image, the GFP fluorescence indicates a similar pattern of the Purkinje Fiber network between mdx and WT mice. Scale bar = 500 µm. Higher magnifications indicated by squares show the normal expression of the gap junction Cx43 at the intercalated discs (IDs) in contractile cardiomyocytes (white arrows) or along the membrane in PF (Yellow arrows) in both mdx and WT mice. Scale bar = 100 µm (WT n = 3; mdx n = 9). (b) Immunofluorescence with Contactin-2, Cx43, DMD and WGA-cy3 antibodies on transversal sections at the mid-ventricular level from WT and mdx mice. While Cntn2 expression, the marker of VCS maturation is similar in WT and mdx mice, DMD is absent in mdx hearts and enriched in their Purkinje fibers compared to other cardiomyocytes in WT. WGA and Cx43 staining at the membrane highlight the cardiac hypertrophy seen in mdx hearts. Scale bar = 50 µm (n = 11).

Article Snippet: Antibodies used in this study were specific to Contactin-2 (AF1714, R&D system), GFP (AbD Serotec), dystrophin (12715-1-AP, ProteinTech), WGA-Cy3 (29076-1, Clinisciences) and WGA-Cy5 (29024-1, Clinisciences).

Techniques: Immunofluorescence, Fluorescence, Expressing, Membrane, Marker, Staining

Journal: Frontiers in Physiology

Article Title: Conduction defects and arrhythmias in mdx mice are not associated with a degeneration of the cardiac Purkinje network

doi: 10.3389/fphys.2025.1607916

Figure Lengend Snippet: Sodium current (I Na ) densities in cardiac Purkinje fibers and ventricular cardiomyocytes from dystrophin-deficient mdx and wild-type (WT) mice at different animal ages. (a) Representative original whole cell I Na traces recorded from Purkinje fibers from 11–13- or 19–21-week-old WT and mdx mice. The pulse protocol to elicit the currents is shown in the inset. (b) Current density-voltage relationships derived from a series of experiments as displayed in a (38 cells, six animals, WT 11–13 weeks; 23 cells, five animals, mdx 11–13 weeks; 48 cells, six animals, WT 19–21 weeks; 34 cells, five animals, mdx 19–21 weeks). The solid lines represent fits with a function given in the methods section. (c) Statistical comparison of current density values at −37 mV between WT and mdx Purkinje fibers from 11–13-week-old mice. In this age range, current densities in mdx Purkinje fibers (−62.6 ± 4.7 pA/pF) were only reduced by trend when compared to WT Purkinje fibers (−74.1 ± 6 pA/pF). (d) Comparison of current density values at −37 mV between WT and mdx Purkinje fibers from 19–21-week-old mice. Here, a significant difference between WT (−72.7 ± 3.3 pA/pF) and mdx (−52.5 ± 3.5 pA/pF) Purkinje fibers existed. (e) I Na densities at −37 mV of ventricular cardiomyocytes of the working myocardium isolated from 16–17-week-old (42 cells from three animals) or 50–51-week-old (38 cells from three animals) mdx mice. There was no significant difference (−15.2 ± 1 pA/pF vs. −14.1 ± 1.1 pA/pF, 16–17 weeks vs. 50–51 weeks). Values represent means ± SE. A nested analysis respecting the hierarchical data structure was used for statistical comparisons .

Article Snippet: Antibodies used in this study were specific to Contactin-2 (AF1714, R&D system), GFP (AbD Serotec), dystrophin (12715-1-AP, ProteinTech), WGA-Cy3 (29076-1, Clinisciences) and WGA-Cy5 (29024-1, Clinisciences).

Techniques: Derivative Assay, Comparison, Isolation

Journal: mBio

Article Title: Staphylococcus aureus Exploits the Host Apoptotic Pathway To Persist during Infection

doi: 10.1128/mBio.02270-19

Figure Lengend Snippet: Caspase-3 is required for deoxyadenosine-induced killing of macrophages. (A) U937-derived macrophages (MΦ) were treated with (+) or without (−) dAdo, and cell lysates were analyzed for caspase-3 activity using a colorimetric assay. As controls, lysates were treated with the caspase-3 inhibitor Ac-DEVD-CHO (+ Inhib.). (B) Survival of MΦ exposed to dAdo and increasing concentrations of Z-DEVD-FMK (0 to 5 μM), an inhibitor of caspase-3. (C) Diagram illustrating the position of CASP3 on chromosome 4 and exons 1 to 8 of CASP3 mRNA. Sequencing results for mutated exon 5 alleles (red box) cloned from CASP3 −/− cells are shown. (D) Immunoblotting of lysates from wild-type (WT) MΦ and their CASP3 −/− and complemented CASP3 −/− variants (+ CASP3 WT ) with caspase-3 and GAPDH-specific antibodies (α-CASP3 and α-GAPDH, respectively). GAPDH was used as a loading control. Numbers to the left of blots indicate the migration of molecular weight markers in kilodaltons. (E and F) Survival of MΦ (black bars) and their SLC29A1 −/− (blue bars), CASP3 −/− (red bars), and complemented CASP3 −/− variants (+ CASP3 WT , white bars) after treatment with dAdo (E) or after treatment with culture medium (RPMI 1640) that had been conditioned by incubation with either wild-type S. aureus Newman or its adsA mutant in the presence of host DNA, as indicated by + and – signs (F). All samples received adenosine deaminase inhibitor (50 μM dCF). Data are the mean (± standard deviation [SD]) values from three independent determinations. Statistically significant differences were analyzed with one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test; ns, not significant ( P > 0.05); **, P < 0.01; ****, P < 0.0001.

Article Snippet: Supernatants were mixed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) loading buffer and boiled at 95°C for 10 min. Proteins were separated on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes for immunoblot analysis with the following rabbit primary antibodies: anti-caspase-3 (anti-CASP3; for U937, antibody ab32351, and for BMDM, antibody ab13847, both from Abcam) and anti-GAPDH (loading control, PA1-987; Thermo Fisher; GAPDH, glyceraldehyde-3-phosphate dehydrogenase).

Techniques: Derivative Assay, Activity Assay, Colorimetric Assay, Inhibition, Sequencing, Clone Assay, Western Blot, Migration, Molecular Weight, Incubation, Mutagenesis, Standard Deviation

Journal: mBio

Article Title: Staphylococcus aureus Exploits the Host Apoptotic Pathway To Persist during Infection

doi: 10.1128/mBio.02270-19

Figure Lengend Snippet: Tissue-specific deletion of caspase-3 impacts S. aureus disease pathogenesis. (A to D) Enumeration of staphylococcal loads (A and C) and visible surface abscesses (B and D) in kidneys after intravenous injection of 10 7 CFU of wild-type S. aureus Newman or its adsA mutant. Data for female (♀ symbol) and male (♂ symbol) animals are displayed separately in panels A and B and in C and D, respectively. Filled (black) circles or bars indicate infection of C57BL/6 CASP3 fl/fl animals; open circles or bars indicate infection of CASP3 fl/fl Tie2-Cre + mice ( n = 10 to 12). Bacterial burden was enumerated as log 10 CFU per kidney at 5 days postinfection. Horizontal blue bars represent the mean CFU count in each cohort (A and C) or indicate the mean (±SD) values of abscesses per kidney (B and D). Data are representative of two independent analyses. Statistically significant differences were analyzed with one-way ANOVA and Tukey’s multiple-comparison test; ns, not significant ( P > 0.05); *, P < 0.05; **, P < 0.01.

Article Snippet: Supernatants were mixed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) loading buffer and boiled at 95°C for 10 min. Proteins were separated on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes for immunoblot analysis with the following rabbit primary antibodies: anti-caspase-3 (anti-CASP3; for U937, antibody ab32351, and for BMDM, antibody ab13847, both from Abcam) and anti-GAPDH (loading control, PA1-987; Thermo Fisher; GAPDH, glyceraldehyde-3-phosphate dehydrogenase).

Techniques: Injection, Mutagenesis, Infection

Journal: mBio

Article Title: Staphylococcus aureus Exploits the Host Apoptotic Pathway To Persist during Infection

doi: 10.1128/mBio.02270-19

Figure Lengend Snippet: Caspase-3 activity suppresses macrophage infiltration into staphylococcal abscesses. (A to P) Immunohistochemical analysis of renal tissues isolated 5 days after intravenous injection of 10 7 CFU of wild-type S. aureus Newman or its adsA mutant into C57BL/6 CASP3 fl/fl or CASP3 fl/fl Tie2-Cre + mice. Thin sections were stained with hematoxylin and eosin (H&E) (A to D) or examined by immunohistochemistry with anti-Ly-6G antibodies (neutrophils) (E to H) or anti-F4/80 antibodies (macrophages) (I to L). (M to P) Magnifications of boxed area from panels I to L. Macrophages and neutrophils stain brown. Green arrows point to replicating S. aureus cells surrounded by a fibrin capsule. Black bars depict a length of 100 μm. Representative images are shown. (Q) Determination of macrophage-infiltrated areas of renal abscesses of infected C57BL/6 CASP3 fl/fl (black circles) or CASP3 fl/fl Tie2-Cre + mice (open circles) by immunohistochemistry with anti-F4/80 antibodies. Macrophage-infiltrated areas were determined by calculating the total and macrophage-free (anti-F4/80-negative) abscess areas. Multiple abscesses ( n = 25 to 35) from a cohort of 3 to 4 animals per group were analyzed. Horizontal blue bars represent mean values in each cohort. (R) Immunoblotting of lysates from bone marrow-derived macrophages (BMDM). Lysates of BMDM derived from female (♀ symbol) or male (♂ symbol) C57BL/6 CASP3 fl/fl or CASP3 fl/fl Tie2-Cre + mice were probed with caspase-3 and GAPDH-specific antibodies (α-CASP3 and α-GAPDH, respectively). GAPDH was used as a loading control. Numbers to the left of blots indicate the migration of molecular weight markers in kilodaltons. (S) Survival of BMDM derived from ♀ and ♂ C57BL/6 CASP3 fl/fl (black bars) or CASP3 fl/fl Tie2-Cre + mice (open bars) after treatment with dAdo and dCF (50 μM). Data are the mean (±SD) values from three independent determinations. Statistically significant differences were analyzed with one-way ANOVA and Tukey’s multiple-comparison test (Q), or by a two-tailed Student's t test (S); ns, not significant ( P > 0.05); **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Supernatants were mixed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) loading buffer and boiled at 95°C for 10 min. Proteins were separated on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes for immunoblot analysis with the following rabbit primary antibodies: anti-caspase-3 (anti-CASP3; for U937, antibody ab32351, and for BMDM, antibody ab13847, both from Abcam) and anti-GAPDH (loading control, PA1-987; Thermo Fisher; GAPDH, glyceraldehyde-3-phosphate dehydrogenase).

Techniques: Activity Assay, Immunohistochemical staining, Isolation, Injection, Mutagenesis, Staining, Immunohistochemistry, Infection, Western Blot, Derivative Assay, Migration, Molecular Weight, Two Tailed Test

Journal: mBio

Article Title: Staphylococcus aureus Exploits the Host Apoptotic Pathway To Persist during Infection

doi: 10.1128/mBio.02270-19

Figure Lengend Snippet: Single nucleotide polymorphisms (SNPs) in CASP3 protect human macrophages from S. aureus -derived deoxyadenosine. (A) Caspase-3 protein lollipop plot highlighting amino acid substitutions investigated in this study. Associated SNP identifiers (IDs) are provided in Table S1 . (B) Immunoblotting of lysates from wild-type (WT) U937-derived macrophages (MΦ) and their SLC29A1 −/− , CASP3 −/− , and complemented CASP3 −/− variants (WT and 12 different alleles indicated according to their amino acid substitution in caspase-3) using caspase-3 and GAPDH-specific antibodies (α-CASP3 and α-GAPDH, respectively). GAPDH was used as a loading control. Numbers to the left of blots indicate the migration of molecular weight markers in kilodaltons. (C) Caspase-3 activity in cell lysates of dAdo-exposed WT MΦ (black bars) and their SLC29A1 −/− (blue bars), CASP3 −/− (red bars), and CASP3 −/− variants complemented with WT (open bars) and various alleles of CASP3 (gray or pink bars). Caspase-3 activity was measured using a colorimetric assay. (D and E) Survival of WT MΦ (black bars) and their SLC29A1 −/− (blue bars), CASP3 −/− (red bars), and CASP3 −/− variants complemented with WT (open bars) and various alleles of CASP3 (gray or pink bars) after treatment with dAdo (D) or after treatment with culture medium (RPMI) that had been conditioned by incubation with either wild-type S. aureus Newman or its adsA mutant in the presence of host DNA, as indicated with + and – signs (E). (C to E) Gray indicates functional CASP3 alleles that support caspase-3 activity, whereas pink depicts nonfunctional alleles that do not restore caspase-3 activity in CASP3 −/− -derived macrophages. All samples received adenosine deaminase inhibitor (50 μM dCF). Data are the mean (±SD) from three independent determinations. Statistically significant differences were analyzed with one-way ANOVA and Tukey’s multiple-comparison test; ns, not significant ( P > 0.05); *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: Supernatants were mixed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) loading buffer and boiled at 95°C for 10 min. Proteins were separated on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes for immunoblot analysis with the following rabbit primary antibodies: anti-caspase-3 (anti-CASP3; for U937, antibody ab32351, and for BMDM, antibody ab13847, both from Abcam) and anti-GAPDH (loading control, PA1-987; Thermo Fisher; GAPDH, glyceraldehyde-3-phosphate dehydrogenase).

Techniques: Derivative Assay, Western Blot, Migration, Molecular Weight, Activity Assay, Colorimetric Assay, Incubation, Mutagenesis, Functional Assay

Journal: mBio

Article Title: Staphylococcus aureus Exploits the Host Apoptotic Pathway To Persist during Infection

doi: 10.1128/mBio.02270-19

Figure Lengend Snippet: Model of macrophage exclusion from staphylococcal abscesses. Diagram illustrating the proposed role of caspase-3 during replication of S. aureus in deep-seated abscesses. Replicating S. aureus cells exploit AdsA to generate dAdo from NETs, thereby triggering caspase-3 activation and macrophage apoptosis. Caspase-3 deficiency promotes macrophage infiltration into infectious foci which affects abscess persistence and prevents the dissemination of bacteria to new foci.

Article Snippet: Supernatants were mixed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) loading buffer and boiled at 95°C for 10 min. Proteins were separated on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes for immunoblot analysis with the following rabbit primary antibodies: anti-caspase-3 (anti-CASP3; for U937, antibody ab32351, and for BMDM, antibody ab13847, both from Abcam) and anti-GAPDH (loading control, PA1-987; Thermo Fisher; GAPDH, glyceraldehyde-3-phosphate dehydrogenase).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to (a) Vcam1 fl/fl Slco1c1-Cre ERT2+/- (Cre+) or Cre ERT2-/- (Cre-) littermates (3-month-old) were treated daily with tamoxifen (i.p. 150 mg/kg) for 5 days followed by 4 days of rest. Mice received 3 LPS injections (0.5 mg/kg i.p.) at 28 hours, 22 hours, and 2 hours prior to perfusion. Mice also received a retro-orbital injection of fluorescently conjugated mouse anti-VCAM1 mAb (100μg) 2 hours prior to perfusion. Representative confocal images of cortex and DG for VCAM1 and Hoechst to label cell nuclei. Loss of Vcam1 in Cre+ mice, but not Cre-, in BBB endothelium, but not in meninges is shown. Scale bar = 100 μm. (b) FACS gating strategy to isolate single BECs. PI+ dead cells were excluded. CD11a/b, CD45, and Ter-119 negative cells were gated to exclude erythrocytes, monocytes/macrophages and microglia. CD13 and ACSA-2 staining was applied to exclude pericytes and astrocytes, respectively. CD31+MECA99+ cells were defined the BEC population. (c) Quantification of (d) flow cytometry that was performed on primary BECs isolated from Cre+ or Cre-mice treated as described in (a). n=3 Cre+ or Cre-mice received LPS, while one Cre-mouse was given PBS vehicle control instead. The VCAM1 gate was set based on a Cre-mice injected with fluorescently conjugated IgG. ***p<0.0007; Unpaired Student’s t-test ; Error bar represents SEM. (e) Experimental Design. n= 6-7 mice/group. (f) Quantification of VCAM1+ percent area in lectin+ vasculature of immunostained sections from 5–6 mice/group. ****p<0.0007; Student’s t-test . (g-i) Quantification of the total number of BrdU+ cells, BrdU+Sox2+ co-labeled neural progenitor cells, and DCX+ immature neurons in the DG of immunostained sections. **p<0.003, Student’s t-test . (j-m) Quantification of Iba1 and CD68 in the DG of immunostained sections. All error bars represent SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLightTM Antibody Labeling Kit (DyLightTM488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Injection, Staining, Flow Cytometry, Isolation, Control, Labeling

Journal: Life Science Alliance

Article Title: Ligand-induced IFNGR1 down-regulation calibrates myeloid cell IFNγ responsiveness

doi: 10.26508/lsa.201900447

Figure Lengend Snippet: (A) IFNGR1 gMFI histograms are plotted from CD14 + PBMCs. The cells were untreated (black), treated with 8 h of 100 U/ml IFNβ (blue), IFNγ (red), or IFNβ and IFNγ together (green). The cells were stained with a biotin-tagged IFNGR1 antibody and treated with streptavidin-APC as a secondary stain, and a histogram of cells treated with secondary antibody alone is also plotted (gray). (B) WT or Ifnar1 −/− BMDCs were stimulated for 0 or 8 h with 100 U/ml IFNβ (blue), IFNγ (red), or IFNβ and IFNγ together (green). Graph depicts gMFI IFNGR1 normalized to those of the respective untreated cells of same genotype. (A, C) WT or Ifngr1 −/− BMDMs were stimulated with 0 or 8 h 100 U/ml IFNγ and stained as in (A). WT untreated (black, “Untx”) histogram shows staining of IFNGR1, whereas Ifngr1 −/− (green) or WT BMDM cells stained with secondary antibody alone (gray) confirmed specificity of the staining. (B, D) Naïve WT splenocytes were treated as in (B). Graph depicts gMFI IFNGR1 on CD90.2+ T cells. (E) WT BMDMs were treated for 8 h ± 100 U/ml recombinant IFNβ, IFNγ, IFNβ, and IFNγ together, IL-6 (10 ng/ml) or IL-10 (50 ng/ml). Graph depicts gMFI IFNGR1 (black bars) or IFNGR2 (gray bars) normalized to untreated cells (gMFI from treated samples/average of gMFI from untreated samples). Unless indicated, statistical significance indicates comparison of IFNGR1 or IFNGR2 to its respective unstimulated (“0 h”) group (dashed line). (F) WT BMDMs were treated for 8 h ± 100 U/ml IFNγ, IL-6 (10 ng/ml), or IL-10 (50 ng/ml). Representative immunoblot depicts lysates probed for pSTAT3Y 705 , Total STAT3, or loading control β-actin. (G) WT BMDMs were treated for indicated amounts of hours (h) with 100 U/ml IFNγ. Graph depicts relative IFNGR1 staining by flow cytometry. (H) WT BMDMs were pretreated for 1 h with mock or with 5 μg/ml JAK inhibitor ruxolitinib (Ruxo) followed by 8 h of 100 U/ml IFNβ (blue) or IFNγ (red). Graph depicts gMFI IFNGR1. (I, J) WT BMDMs were heated at 60°C for 5 min (heat killed) or treated for 0 or 8 h with 100 U/ml IFNβ, IFNγ, or IFNβ and IFNγ together. In additional combination treatments, either 100 U/ml IFNβ or IFNγ was combined with 10 or 1,000 U/ml of the other cytokine. (I, J) Graphs depict (I) % live cells or (J) gMFI IFNGR1 gated on live, CD11b+ cells. For each panel, bar graphs represent the mean ± SD of the pooled values for each condition (n = 3 independent experiments). Error bars represent SEM; n.s., * P < 0.05, ** P < 0.001, ** P < 0.001 by one-way ANOVA and Dunnett’s or Tukey’s post-hoc test for comparison between unstimulated (“0 h,” “Untx,” “Mock,” dashed line) and other groups or comparison between multiple conditions. n.s., not significant.

Article Snippet: For inhibition of JAK, WT BMDMs were treated with 5 μg/ml ruxolitinib (#S1378; Selleckchem) for 1 h. To inhibit transcription, the cells were treated with 1 μg/ml actinomycin D (#AC29494-0050; Thermo Fisher Scientific).

Techniques: Staining, Recombinant, Comparison, Western Blot, Control, Flow Cytometry

Journal: Life Science Alliance

Article Title: Ligand-induced IFNGR1 down-regulation calibrates myeloid cell IFNγ responsiveness

doi: 10.26508/lsa.201900447

Figure Lengend Snippet: (A) WT or Ifnar1 −/− BMDMs were stimulated with 0 or 8 h 100 U/ml IFNβ (blue), IFNγ (red), or IFNβ and IFNγ together (green). The cells were lysed and probed for immunoblot. Graph depicts density of IFNGR1 bands normalized to β-actin (n = 3 independent experiments). (B) WT BMDMs were treated for 8 h ± 100 U/ml recombinant IFNβ, IFNγ, IFNβ, and IFNγ together, IL-6 (10 ng/ml) or IL-10 (50 ng/ml). Total RNA was isolated and analyzed by qRT-PCR. The relative transcript abundance of Ifngr1 (black bars) or Ifngr2 (gray bars) was calculated using the 2 −ΔΔCt model with GAPDH mRNA as a housekeeping gene. (B, C) WT BMDMs were treated for 0, 2, 4, and 6 h with 100 U/ml IFNγ (red bars) or 1 μg/ml actinomycin D (gray bars). Total RNA was isolated and relative Ifngr1 transcript abundance analyzed by qRT-PCR as in (B). For each panel, bar graphs represent the mean ± SD of the pooled values for each condition (n = 3 independent experiments). Error bars represent SEM; n.s., * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA and Dunnett’s or Tukey’s post-hoc test for comparison between untreated (“0 h,” “Mock,” dashed line); and other groups or comparison between conditions. n.s., not significant.

Article Snippet: For inhibition of JAK, WT BMDMs were treated with 5 μg/ml ruxolitinib (#S1378; Selleckchem) for 1 h. To inhibit transcription, the cells were treated with 1 μg/ml actinomycin D (#AC29494-0050; Thermo Fisher Scientific).

Techniques: Western Blot, Recombinant, Isolation, Quantitative RT-PCR, Comparison

Journal: Life Science Alliance

Article Title: Ligand-induced IFNGR1 down-regulation calibrates myeloid cell IFNγ responsiveness

doi: 10.26508/lsa.201900447

Figure Lengend Snippet: WT or Ifnar1 −/− BMDMs or RAW 264.7 reporter cells were treated 0–8 h with 100 U/ml recombinant IFNβ (blue) or IFNγ (red). (A, B) Total RNA was isolated and analyzed by qRT-PCR. The relative transcript abundance of Ifngr1 was calculated using the 2 −ΔΔCt model with Gapdh and Hmbs mRNA as housekeeping genes. (C) ChIP was performed for pS5-RNA pol II in WT BMDMs. Murine primers that amplify 100 base pairs within exon 1 of I fngr1 were used to quantify immunoprecipitated chromatin by qRT-PCR. (D) Luciferase activity from lysates of RAW 264.7 reporter cells with luciferase driven by the proximal Ifngr1 promoter. Luciferase activity values were normalized to those of the respective untreated cells. Values were derived from three separate experiments using two independently transfected Ifngr1 promoter-luciferase cell lines. Statistical significance indicates comparison of relative light units between unstimulated (“0 h”; dashed line) and stimulated groups by one-way ANOVA and Dunnett’s post hoc test. (C, E) WT BMDMs were treated as in (C). ChIP assays were performed for Egr3. Murine primers that amplify an Egr site in the proximal Ifngr1 promoter were used to quantify immunoprecipitated chromatin by qPCR. (C, F) Human THP-1 cells were treated as in (C). ChIP assays were performed for H3K4me3. Human primers that amplify putative enhancer regions upstream of the Ifngr1 promoter were used to quantify H3K4me3-associated chromatin by qPCR. (C, E, F) Graphs depict fold enrichment over isotype values normalized to those of the respective unstimulated cells (relative fold enrichment over isotype = fold enrichment from treated sample/average fold enrichment from untreated sample). (G) Naïve WT and fGR1 splenic monocytes were treated for 8 h ± 100 U/ml recombinant IFNβ (blue) or IFNγ (red). Graph depicts relative gMFI IFNGR1 on gated splenic monocytes. For each panel, bar graph represents the mean ± SD of pooled values per condition (n = 3 independent experiments); n.s., * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA and Dunnett’s or Tukey’s post-hoc test for comparison between untreated and other groups or comparison between conditions. n.s., not significant.

Article Snippet: For inhibition of JAK, WT BMDMs were treated with 5 μg/ml ruxolitinib (#S1378; Selleckchem) for 1 h. To inhibit transcription, the cells were treated with 1 μg/ml actinomycin D (#AC29494-0050; Thermo Fisher Scientific).

Techniques: Recombinant, Isolation, Quantitative RT-PCR, Immunoprecipitation, Luciferase, Activity Assay, Derivative Assay, Transfection, Comparison

Journal: Life Science Alliance

Article Title: Ligand-induced IFNGR1 down-regulation calibrates myeloid cell IFNγ responsiveness

doi: 10.26508/lsa.201900447

Figure Lengend Snippet: WT BMDMs or fGR1 peritoneal macrophages were stimulated and lysed. Representative immunoblots depict lysates probed for pSTAT1Y 701 , Total STAT1, and β-actin. (A) WT BMDMs were treated for 30 min with 100 U/ml IFNγ, washed with PBS, and rested for 0, 0.5, 1, 1.5, 2, 3, 4, or 5 h in cytokine-free media. (B) Top row depicts WT BMDM lysates probed at 5, 15, 30, or 60 min post stimulation with IFNγ with no pretreatment (“Untx,” right side). Second row depicts pSTAT1Y 701 from the following treatments: lane 1 (L1), untreated (“Untx”); L2, 30 min (30′) IFNγ; L3–L7, pretreated with 30′ IFNγ “pulse,” PBS wash, 5 h rest in cytokine-free medium followed by secondary stimulation “hit” of 100 U/ml IFNγ for 0, 5, 15, 30, or 60 min. (C) WT BMDM lysates probed from the following treatments: lane 1 (L1), untreated (“Untx”); L2, 30 min (30′) IFNγ; L3, pretreated with 30′ IFNγ “pulse,” PBS wash, 5 h rest in cytokine-free media; L4, as in L3 followed by secondary “hit” of 30′ 100 U/ml IFNγ; L5, as in L3 followed by secondary “hit” of 30′ 100 U/ml IFNβ. (D) WT BMDM lysates probed from the following treatments: lane 1 (L1), untreated (“Untx”); L2, 30 min (30′) IFNγ; L3, pretreated with 30′ IFNγ “pulse,” PBS wash, 5 h rest in cytokine-free medium; L4, as in L3 followed by secondary “hit” of 30′ 100 U/ml IFNγ; L5, pretreated with 30′ IFNγ “pulse,” PBS wash, 12 h rest in cytokine-free medium; L6, as in L5 followed by secondary “hit” of 30′ 100 U/ml IFNγ. (E) fGR1 peritoneal macrophages probed from the following treatments: lane 1 (L1), untreated (“Untx”); L2, 30 min (30′) IFNγ; L3, pretreated with 30′ IFNγ “pulse,” PBS wash, 5 h rest in cytokine-free medium; L4, as in L3 followed by secondary “hit” of 30′ 100 U/ml IFNγ. (F) Top row depicts WT BMDM lysates probed for pSTAT1Y 701 from the following treatments: lane 1 (L1), untreated (“Untx”); L2, 30 min (30′) 100 U/ml IFNγ; L3, pretreated with 100 U/ml 30′ IFNγ “pulse,” PBS wash, 5 h rest in cytokine-free media; L4–L9, as in L3 followed by 30′ secondary “hit” with increasing concentrations of IFNγ: 10, 100, 300, 500, 1,000, or 2,000 U/ml. For each panel, error bars represent SEM. Each bar graph depicts density of pSTAT1Y 701 bands normalized to β-actin (n = 3 independent experiments). (D, E) Each line graph depicts relative gMFI IFNGR1; n.s., *** P < 0.001 by one-way ANOVA and Dunnett’s or Tukey’s post-hoc test for comparison between untreated and other groups or comparison between conditions. n.s., not significant. Source data are available for this figure.

Article Snippet: For inhibition of JAK, WT BMDMs were treated with 5 μg/ml ruxolitinib (#S1378; Selleckchem) for 1 h. To inhibit transcription, the cells were treated with 1 μg/ml actinomycin D (#AC29494-0050; Thermo Fisher Scientific).

Techniques: Western Blot, Comparison

Journal: Life Science Alliance

Article Title: Ligand-induced IFNGR1 down-regulation calibrates myeloid cell IFNγ responsiveness

doi: 10.26508/lsa.201900447

Figure Lengend Snippet: (A, B) Graphs depict density of pSTAT1Y 701 bands normalized to β-actin (n = 3 independent experiments) correlating with representative immunoblot from (A) or (B) . (C, D, E) WT BMDMs or peritoneal macrophages were stimulated and lysed. Representative immunoblots depict lysates probed for (C and E) pSTAT3Y 705 , Total STAT3, and β-actin, or (D) pSTAT1Y 701 , Total STAT1, and β-actin. (C) WT BMDM lysates probed from the following treatments: lane 1 (L1), untreated (“Untx”); L2, 30 min (30′) IFNγ; L3, pretreated with 30′ IFNγ “pulse,” PBS wash, 5 h Rest in cytokine-free medium; L4, as in L3 followed by secondary “hit” of 30′ 100 U/ml IFNγ; L5, as in L3 followed by secondary “hit” of 30′ 100 U/ml IFNβ. (D) WT peritoneal macrophages probed from the following treatments: lane 1 (L1), untreated (“Untx”); L2, 30 min (30′) IFNγ; L3, pretreated with 30′ IFNγ “pulse,” PBS wash, 5 h Rest in cytokine-free medium; L4, as in L3 followed by secondary “hit” of 30′ 100 U/ml IFNγ. (E) Top row depicts WT BMDM lysates probed for pSTAT3Y 705 from the following treatments: lane 1 (L1), untreated (“Untx”); L2, 30 min (30′) 100 U/ml IFNγ; L3, pretreated with 100 U/ml 30′ IFNγ “pulse,” PBS wash, 5 h Rest in cytokine-free medium; L4–L9, as in L3 followed by 30′ secondary “hit” with increasing concentrations of IFNγ: 10, 100, 300, 500, 1,000, or 2,000 U/ml. For each panel, error bars represent SEM. (A, B, C, D, E) Each bar graph depicts density of (A, B, and D) pSTAT1Y 701 or (C and E) pSTAT3Y 705 bands normalized to β-actin (n = 3 independent experiments); (D) Line graph depicts relative gMFI IFNGR1; n.s., *** P < 0.001 by one-way ANOVA and Dunnett’s or Tukey’s post-hoc test for comparison with unstimulated (“Untx”; dashed line) or between multiple conditions, respectively. n.s., not significant. Source data are available for this figure.

Article Snippet: For inhibition of JAK, WT BMDMs were treated with 5 μg/ml ruxolitinib (#S1378; Selleckchem) for 1 h. To inhibit transcription, the cells were treated with 1 μg/ml actinomycin D (#AC29494-0050; Thermo Fisher Scientific).

Techniques: Western Blot, Comparison