Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Phagolysosomes do not undergo exocytosis but fragment. (A) 1 h after phagocytosis of IgG-opsonized beads, RAW cells were treated with 10 µM ionomycin in the presence of 1.2 mM of Ca 2+ , and live-cell DIC images were acquired at the indicated time points. Scale bars: 10 µm. (B) Number of latex beads per cell at the indicated treatment times, normalized to the number of beads/cell observed at the onset of the treatment (T 0 ). Data are mean ± SEM of 3 independent experiments, where 10 cells were measured in each experiment. (C) RAW cells expressing PLCδ1-PH-GFP and containing IgG-opsonized beads in phagosomes were treated with 10 µM ionomycin or DMSO (control) in the presence of 1.2 mM of Ca 2+ . Images were acquired before or after 10 min of treatment. Asterisks correspond to the positions of the beads, as shown in the insets. Scale bars: 10 µm. (D) Live-cell imaging time series of IgG-aggregated fluorescent 0.1-µm latex beads after 2-h phagocytosis by RAW cells. Beads are depicted in a rainbow scale. Red and blue correspond to the highest and lowest florescence intensity levels, respectively. The smaller panels show the far red and DIC channels for the first frame. The cell contour is delineated with white dashes. See corresponding . Scale bars: 5 µm. (E) Macrophages were assessed for exocytosis of digested phagosomal contents. RAW macrophages internalized mRFP1-labeled E. coli for 1 h and then were incubated for the indicated time points. TCA precipitates of cell media and cell lysates were probed for mRFP1, detecting a native and a cleaved mRFP1 product that accumulated over time. GAPDH was used as a loading control and to detect cell lysis. (F) Quantification of cleaved mRFP1 levels normalized to GAPDH in media and within cells. Data are shown as mean ± SD of six independent experiments. Conditions were compared using one-way ANOVA and Holm-Sidak’s post hoc test (*, P < 0.05).

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Expressing, Control, Live Cell Imaging, Labeling, Incubation, Lysis

Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Fragmentation of the phagolysosome requires cargo degradation and the cytoskeleton. (A, C, E, and G) Macrophages engulfed E. coli for 15 min, chased for 25 min, and treated with either Con A and NH 4 Cl (A), protease inhibitor cocktail (C), microtubule inhibitors (E), actin inhibitors (G), or vehicle control (DMSO). Cells were fixed after 1 or 4 h after phagocytosis and stained with an anti– E. coli antibody whose fluorescence is displayed in rainbow scale. Dashed lines show cell contours. Scale bars: 5 µm. (B, D, F, and H) Total volume of PDVs per cell. Data are mean ± SEM of 3 independent experiments with 25 cells quantified per condition of an experiment and compared by one-way ANOVA with Tukey’s test. Conditions labeled with different letters (a–d) are statistically different (P < 0.05).

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Protease Inhibitor, Control, Staining, Fluorescence, Labeling

Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Clathrin is necessary for the resolution of the phagolysosome. (A) Imaging of RAW cells 4 h after phagocytosis of mCherry- Lp (rainbow scale). Pitstop was added 10 min before imaging. See corresponding . Scale bars: 5 µm. (B) Quantification of the volume of phagosomes and PDVs over time. Volumes were normalized to T 0 for each treatment. Data are mean ± SEM of three independent experiments. *, P < 0.05 indicates that the regressions are significantly different. (C) RAW cells engulfed mRFP1– E. coli for 15 min, chased for 25 min, and treated with Pitstop or ikarugamycin. Cells were fixed 1 or 4 h after phagocytosis and immunostained against E. coli (rainbow scale). Scale bars: 5 µm. (D) Volume of PDVs per cell for experiments shown in C. Data are mean ± SEM of 3 independent experiments with 25 cells quantified per condition of an experiment, and results were tested by one-way ANOVA with Tukey’s test. Different letters indicate results are statistically different (P < 0.05). (E) RAW cells expressing Mito-mCherry-FRB and GFP-FKBP-CLC were treated with rapamycin to induce knocksideways (KSW) of the clathrin light chain or DMSO (Ctrl) and allowed to internalize E. coli . Cells were fixed 3 h after phagocytosis, stained for E. coli , and imaged. Scale bars: 10 µm. (F) Number of fragments stained with anti– E. coli antibodies in control and rapamycin-treated cells. Data are mean ± SEM of three independent experiments and statistically tested using an unpaired t test (*, P < 0.05). Dashed lines show cell contours (A, C, and E).

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Imaging, Expressing, Staining, Control

Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Dynamin inhibition blocks phagosome fragmentation. (A and C) 40 min after phagocytosis of mRFP1– E. coli , RAW cells were treated with the dynamin inhibitors dyngo-4a, dynole 34-2, or vehicle (DMSO); fixed at the indicated time points; and immunostained for E. coli . E. coli fluorescence labeling is shown in rainbow scale. Red and blue are the highest and lowest intensity levels, respectively. Dashed lines indicate the boundary of the cells. Scale bars: 5 µm. (B and D) The total volume of PDVs per cell for the indicated treatments. Data are mean ± SEM of sample of 25 cells from 3 independent experiments. Treatments were compared statistically using a one-way ANOVA with Tukey’s post hoc test. Conditions with different letters (a–c) indicate statistically significant difference (P < 0.05). (E) Macrophages treated with dynole 34-2 or DMSO (vehicle) internalized Alexa Fluor 546–labeled transferrin for 10 min before fixation. White dashes indicate cell boundaries. Scale bars: 30 µm.

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Inhibition, Fluorescence, Labeling

Journal: Immunology

Article Title: IL‐3 is essential for ICOS‐L stabilization on mast cells, and sustains the IL‐33‐induced RORγt + T reg generation via enhanced IL‐6 induction

doi: 10.1111/imm.13305

Figure Lengend Snippet: IL‐3 stabilizes ICOS‐L on mast cells via ERK signalling. Bone marrow‐derived mast cells (BMMCs) were cultured as indicated, and ICOS‐L expression was assessed on CD117 + BMMCs by flow cytometry. (A‐C) BMMCs were generated from total bone marrow cells of C57BL/6 mice in medium containing supernatant of a X63Ag‐653 BPV‐rmIL‐3 stably transfected cell line equivalent to 20 ng/ml IL‐3. Differentiation was monitored over 8 weeks ( n = 12 independent biological replicates). FACS plots in (A) show representative results after 1–2 weeks or after 7–8 weeks of BMMC generation. Generation of CD117 + BMMCs is summarized in (B). ICOS‐L expression among CD117 + BMMCs is summarized in (C). (D) BMMCs were cultured in the presence of supernatant of a X63Ag‐653 BPV‐rmIL‐3 stably transfected cell line equivalent to 20 ng/ml IL‐3 with or without a blocking anti‐IL‐3 antibody (10 µg/ml) for 24 h. CD117 + BMMCs and ICOS‐L + cells among CD117+ BMMCs are summarized in the diagrams ( n = 6 independent biological replicates). (E) BMMCs were cultured in medium alone or medium containing either 50 ng/ml recombinant IL‐3, 50 ng/ml recombinant IL‐33 or 50 ng/ml recombinant SCF for 24 h. Frequencies of ICOS‐L + cells among CD117 + BMMCs are summarized ( n = 3 independent biological replicates). (F‐I) BMMCs were starved from IL‐3 for 60‐min prior stimulation with 50 ng/ml IL‐3 for the indicated time periods. Phosphorylation of MAP kinases ERK1/2, JNK1/2 and p38 was analysed by Western blotting. Representative Western blot bands are shown in (F). Phosphorylation of MAP kinases ERK1/2 (G), p38 (H) and JNK1/2 (I) was quantified ( n = 4 biologically independent experiments). (J) BMMCs were cultured in the presence of recombinant IL‐3 and DMSO or with JNK inhibitor (SP600125, n = 3 biological replicates), p38 inhibitor (SB203580, n = 6 biological replicates), or ERK inhibitor (UO126, n = 6 biological replicates) for 24 h. Subsequently, frequencies of ICOS‐L + cells among CD117 + BMMCs were analysed and summarized in the diagram. (K) BMMCs were starved from IL‐3 for 30 min and incubated in the presence of DMSO or UO126 for further 30 min. Subsequently, cells were cultured in medium alone or in the presence of 50 ng/ml IL‐3 for 24 h. Phosphorylation of MAP kinases ERK1/2 was analysed by Western blotting. Representative Western blot bands are shown, and quantification of ERK phosphorylation is summarized in the diagram below ( n = 3 independent biological replicates). Diagrams show average values of all experiments ± SEM. Statistics were done with Student's t‐ test: n.s . not significant; * p < 0.05; ** p < 0.01, *** p < 0.001

Article Snippet: After 1 h, BMMCs were preincubated with DMSO (vehicle) or inhibitors (all from Merck) for 30 min.

Techniques: Derivative Assay, Cell Culture, Expressing, Flow Cytometry, Generated, Stable Transfection, Transfection, Blocking Assay, Recombinant, Western Blot, Incubation

Journal: Cancers

Article Title: CYP26A1 Links WNT and Retinoic Acid Signaling: A Target to Differentiate ALDH+ Stem Cells in APC -Mutant CRC

doi: 10.3390/cancers16020264

Figure Lengend Snippet: Wt-APC expression sensitizes HT29 cells to ATRA’s ability to decrease cell proliferation and attenuates ATRA-induced WNT/β-catenin activity. ( A ) Cell proliferation was reduced by 65% when wt-APC was induced by ZnCl 2 and cells were treated with 20 µM ATRA for 48 h. Values are normalized to mt-APC DMSO control. ( B ) Cell proliferation was reduced by approximately 10% (10 μM) and 20% (20 μM) in mutant- APC ( mt-APC ) cells (black line). Cell proliferation was further reduced by approximately 36% (10 μM) and 42% (20 μM) in wild-type APC ( wt-APC ) cells (dashed gray line). Similar results were seen at 24 h, altogether indicating a time- and concentration- dependent effect. Mt-APC and wt-APC values were normalized to each of their respective DMSO controls. ( C ) TCF/LEF reporter assay was performed to determine WNT/β-catenin activity. ATRA caused an increase in WNT activity in mt- APC cells (black bars). However, this increase was partially attenuated when wt-APC expression was induced (gray bars). Experiments ( n = 3) were performed with error bars plotted as standard error of the mean (SEM). Multiple unpaired t -test was used to determine statistical significance at p < 0.05.

Article Snippet: Controls involved no ZnCl 2 or DMSO (0.1%; Fisher Scientific, Waltham, MA, USA) vehicle.

Techniques: Expressing, Activity Assay, Control, Mutagenesis, Concentration Assay, Reporter Assay