Journal: Nature Communications

Article Title: Facilitative lysosomal transport of bile acids alleviates ER stress in mouse hematopoietic precursors

doi: 10.1038/s41467-021-21451-6

Figure Lengend Snippet: a Quantification of blood parameters (red blood cell count (RBC), hemoglobin (Hb), and hematocrit) from 2 to 16- week-old Slc29a3 +/+ (black) and Slc29a3 −/− (red) mice. Data represent mean ± SEM ( n = 6 mice/group, * p < 0.05 by two-way ANOVA and Tukey’s post hoc test). b Representative images of femurs and spleens excised from 2 to 16-week-old Slc29a3 +/+ and Slc29a3 −/− mice; Scale bar 2 mm. c Cellularity of excised femurs and spleens in Slc29a3 +/+ (black) and Slc29a3 −/− (red) mice. Data represent mean ± SEM ( n = 6 mice/group, * p < 0.05 by two-way ANOVA and Tukey’s post hoc test). d Representative H&E stained bone marrow (×10; Scale bar = 100 mm) and splenic sections (×4; Scale bar = 200 mm) comparing Slc29a3 +/+ with Slc29a3 −/− mice at 16 weeks. One representative image from three independent experiments is shown for the bone marrow and spleen. Insets show higher magnification (×40; Scale bar = 20 mm) of boxed regions. Note, bone marrow is severely hypocellular with hematopoietic cell loss and erythroid progenitor cell death (green arrows) in Slc29a3 −/− mice. Also, severe atrophy of splenic WP with diffuse expansion of the RP with signs of hematopoiesis and erythroid cell death (green arrows). WP white pulp, RP red pulp. e Flow cytometry quantification of erythroid cell composition of bone marrow (8 weeks) and spleen (16 weeks) in Slc29a3 +/+ ( black ) and Slc29a3 −/− ( red ) mice. Data represent mean ± SEM ( n = 6 mice/group, * p < 0.05 by two-tailed t -test). f Plasma erythropoietin EPO levels in Slc29a3 +/+ (black) and Slc29a3 −/− (red) mice from 2 to 16-week-old. Data represent mean ± SEM ( n = 4 mice/group, * p < 0.05 by two-tailed t -test). g Survival time, Hb%, HCT%, bone marrow cellularity, and BFU-E recovery after 6.5 Gy total body irradiation (TBI) of Slc29a3 −/− (red) mice at 8 weeks old. Data represent mean ± SEM ( n = 6 mice/group, * p < 0.05 by two-tailed t -test). Survival curves were plotted with the Kaplan–Meier method ( n = 6/group, *** P < 0.001; Mantel-Cox test). h LSK, LSK Flt3 - , ST-HSCs, and LT-HSCs were examined for relative cell number in 8-week-old Slc29a3 +/+ mice pre (day 0) (gray) and post (day 15) (black) irradiation, and Slc29a3 −/− mice pre (day 0) (blue) and post (day 15) (red) irradiation. Data represent mean ± SEM ( n = 6 mice/group, * p < 0.05 by ANOVA and Tukey’s post hoc test).

Article Snippet: Alternative conjugates for CD34 (Alexa Fluor 700-anti-CD34, clone RAM34; 1:50 dilution), IL7Ra (PE-CF594-anti-CD127, clone SB/199; 1:50 dilution), and Flt3 (APC-anti-CD135, clone A2F10.1; 1:50 dilution) were also obtained from BD Biosciences (San Jose, CA, USA).

Techniques: Cell Counting, Staining, Flow Cytometry, Two Tailed Test, Irradiation

Journal: Nature Communications

Article Title: Facilitative lysosomal transport of bile acids alleviates ER stress in mouse hematopoietic precursors

doi: 10.1038/s41467-021-21451-6

Figure Lengend Snippet: a LSK, LSK Flt3 - , ST-HSCs, and LT-HSCs were examined for the aggresomal marker Thioflavin T (TFT), ER stress markers (GRP78, GRP94, p-PERK, and p-eI2Fα), reactive oxygen species (ROS), and apoptotic markers (annexin V, and cleaved caspase-3) in 12-week-old Slc29a3 +/+ mice pre (day 0) (gray) and post (day 15) (black) irradiation, and Slc29a3 −/− mice pre (day 0) (blue) and post (day 15) (red) irradiation. Data represent mean ± SEM normalized to Slc29a3 +/+ pre (day 0) and post (day 15) irradiation ( n = 6 mice/group, except cleaved caspase 3 and annexin V n = 3 mice/group, * p < 0.05 by ANOVA and Tukey’s post hoc test). b The proportion of LT-HSCs in quiescence (G 0 ) or expressing FOXO3a in 8-week-old Slc29a3 +/+ mice pre (day 0) (gray) and post (day 15) (black) irradiation, and Slc29a3 −/− mice pre (day 0) (blue) and post (day 15) (red) irradiation. Data represent mean ± SEM ( n = 6 mice/group, * p < 0.05 by two-tailed t -test). c CHOP/GRP78 ratio in LT-HSCs in 8-week-old Slc29a3 +/+ (black) and Slc29a3 −/− (red) mice pre (day 0) and post (day 15) irradiation. Data represent mean ± SEM ( n = 6 mice/group, * p < 0.05 by two-tailed t -test). d Treatment with the PERK inhibitor (GSK2606414, 0.5 mM) (blue) or without (gray) alters cell death in Slc29a3 −/− HSPCs in the presence of tunicamycin (0–2 mg/ml) or thapsigargin (0–2 mM). Data represent mean ± SEM ( n = 6 mice/group, * p < 0.05 by two-tailed t -test). e A proportion of LT-HSCs exhibiting high Thioflavin T fluorescence (ThT high ) and ROS positivity (ROS + ) when cultured in the presence of DMSO (vehicle; red) or salubrinal (10 mM; blue). Data represent mean ± SEM ( n = 6, * p < 0.05 by two-tailed t -test). f Proportion of LT-HSCs positive for FOXO3a or cCasp3 when cultured in the presence of DMSO (vehicle; red) or salubrinal (10 mM; blue). Data represent mean ± SEM ( n = 6, * p < 0.05 by two-tailed t -test). g Relative number of HSPCs and their subsets positive for GRP78 and ROS in Slc29a3 +/+ and Slc29a3 −/− bone marrow after 6.5 Gy TBI followed by daily treatment vehicle (DMSO; red) or salubrinal (gray; 1 mg/kg, s.c.) and fold change of HSPCs and erythroid cells after 6.5 Gy TBI followed by daily treatment vehicle (DMSO) (red) or salubrinal (gray) (1 mg/kg, s.c.). Data represent mean ± SEM ( n = 6, * p < 0.05 by two-tailed t -test). h Survival curves for 60 days were plotted with the Kaplan–Meier method ( n = 6/group, *** p < 0.001; Mantel-Cox test). Vehicle (DMSO; red); Sal salubrinal (1 mg/kg, s.c; gray), 4-PBA 4-phenylbutyrate (10 mg/kg, s.c.; blue).

Article Snippet: Alternative conjugates for CD34 (Alexa Fluor 700-anti-CD34, clone RAM34; 1:50 dilution), IL7Ra (PE-CF594-anti-CD127, clone SB/199; 1:50 dilution), and Flt3 (APC-anti-CD135, clone A2F10.1; 1:50 dilution) were also obtained from BD Biosciences (San Jose, CA, USA).

Techniques: Marker, Irradiation, Expressing, Two Tailed Test, Fluorescence, Cell Culture

Journal: Immunity

Article Title: Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication

doi: 10.1016/j.immuni.2022.05.013

Figure Lengend Snippet:

Article Snippet: PeCF594 rat anti-mouse CD135 (clone: A2F10.1) , BD Bioscience , Cat#: 562537; RRID: AB_2737639.

Techniques: Produced, Subcloning, Recombinant, Electron Microscopy, Protease Inhibitor, Purification, Modification, Selection, Western Blot, In Situ, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Bradford Protein Assay, Mouse Assay, Sequencing, Real-time Polymerase Chain Reaction, Clone Assay, Software