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: Frontiers in Immunology

Article Title: Plasmodium curtails autoimmune nephritis via lasting bone marrow alterations, independent of hemozoin accumulation

doi: 10.3389/fimmu.2023.1192819

Figure Lengend Snippet: P. yoelii (Py) infection and transfer of BM from previously infected mice protects from kidney nephritis without reducing autoreactivity. (A) Schematic representation of the experimental injections performed. To ensure equivalent infection rates, seeder R2 mice were injected with frozen aliquots of Py -infected or non-infected RBCs. At a point when parasitemia is 4% (usually 8 days after injection), the amount of blood equivalent to one million infected RBCs was injected i.p into experimental mice. Control mice received the same amount of blood from uninfected mice. After the infection is cleared, long-term lupus symptoms were measured (autoantibodies and kidney nephritis). For BM transfer, we extracted BM cells from R2 mice that were infected or uninfected (1-month post-infection, without presence of parasite) and transferred into R2 mice. (B) Representative images of IgG serum autoantibodies using the ANA test. WT refers to C57Bl/6J mice. Serum from experimental mice was incubated over Hep-2 cells and then stained with Goat anti-mouse IgG Alexa 488. Scale bar 50 µm. (C) Anti-nuclear antibodies (ANA) in serum 4 months after transfer. Scores were defined by the positivity for different serum dilutions: ANA 1 (1:100), ANA 2 (1:300), ANA 3 (1:900), ANA 4 (1:2700), ANA 5 (1:8100). Positive (score 5, R2 mice) and negative (Score 0, WT mice) controls were used to define the scores. Data are represented as mean for 5 mice per group, n.s; non-significant. (D) Representative images of kidney sections stained with Hematoxylin and Eosin (H&E) autoantibodies from 3 independent experiments. Glomerulus (white stars), infiltrated areas (dashed black lines) and protein deposits (black arrowheads) are indicated. Scale bar 100 µm. (E) NIH activity and chronicity indexes calculated as described in methods section. Detailed scoring shown in Figure S1 . Data are represented as the mean ± SD from 5 mice per group. Student t-test *p<0.05, **p<0.01, ***p<0.001. (F) Scheme showing various populations in the BM DC lineage. Markers used for identification of individual populations by flow cytometry were colored in green (lineage markers) or in red (gating strategy). Common DC precursors (CDP, c-Kit lo ) were identified by the lack of lineage markers (Ter119, CD11b, CD11c, Nkp46, CD3, CD19 and Gr1) and the expression of CX3CR1, CD115 and CD135. Conventional DC (cDC) stages were distinguished by the high (Pre-cDCs) or low (cDCs) expression of MHC-II among the CD11c + Gr1 - F4/80 - population. (G) Graphical representation of frequencies BM populations quantified by flow cytometry in WT and R2 mice 4 months after infection. Data are representative of two separate experiments, one of them shown indicated in the graph as the mean ± SD from 2 (WT Ctrl), 3 (WT Py), 9 (R2 Ctrl) and more than 6 (R2 Py) mice per group. Pair comparison analysis denoted no significant differences between infected and uninfected samples in each genotype group.

Article Snippet: DC precursors were gated using the following antibodies: CX3CR1-PE (clone SA011F11, Biolegend), SiglecH-BV421 (clone 551, Biolegend), CD115-APC (clone AFS98, Biolegend), CD135-PE (clone A2F10.1, BD Biosciences), Ly6-G/Ly6-C (Gr-1)-APC-Cy7 (clone RB6-8C5, Biolegend), F4/80-APC (clone BM8, Biolegend).

Techniques: Infection, Injection, Incubation, Staining, Activity Assay, Flow Cytometry, Expressing

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

Journal: Scientific Data

Article Title: Short-read and long-read RNA sequencing of mouse hematopoietic stem cells at bulk and single-cell levels

doi: 10.1038/s41597-021-01078-4

Figure Lengend Snippet: Sample collection and workflow. ( a ) Sample collection and sequencing methods. Bone marrow cells (BMC) were isolated, and lineage negative (LIN − ) cells were enriched using magnetic beads. Long-term (LT) and short-term (ST) hematopoietic stem cells (HSC) and multipotent progenitors (MPP) were sorted according to their surface makers: LT-HSC (Sca-1 + c-Kit + CD34 − CD135 − ), ST-HSC (Sca-1 + c-Kit + CD34 + CD135 − ) and MPP (Sca-1 + c-Kit + CD34 + CD135 + ). Single-cell or 100 cells (bulk P100) were sorted for library construction following the Smart-seq2 protocol. The cDNA libraries were used for short-read (Illumina Hiseq) or long-read (Nanopore or PacBio) sequencing. ( b ) Gating strategy for cell sorting. The main population was gated via FSC-A (forward scatter) and SSC-A (side scatter), and single cells were gated via FSC-A and FSC-H. FSC, forward scatter; SSC, side scatter; A, area; H, height.

Article Snippet: The following antibodies were used for staining (all purchased from BD): PerCP/Cyanine5.5 anti-CD11b Antibody (Clone M1/70, Cat No. 550993), PerCP/Cyanine5.5 anti-CD3e (Clone 145-2C11, Cat No. 551163), PerCP/Cyanine5.5 anti-TER119 (Clone TER119, Cat No. 560512), PerCP/Cyanine5.5 anti-Gr-1 (Clone RB6-8C5, Cat No. 552093), PerCP/Cyanine5.5 anti-B220 (Clone RA3-6B2, Cat No. 552771), BV421 anti-CD135 (Clone A2F10.1, Cat No. 562898), PE/Cyanine7 anti-Sca-1 (Clone D7, Cat No. 558162), APC/Cyanine7 anti-CD117 (c-Kit) (Clone 2B8, Cat No. 560185), FITC anti-CD34 (Clone RAM34, Cat No. 553733), Fixable Viability Stain 510 (BD Pharmingen, Cat No. 564406).

Techniques: Sequencing, Isolation, Magnetic Beads, FACS

Journal: Journal of Cellular and Molecular Medicine

Article Title: Telocytes transfer extracellular vesicles loaded with microRNAs to stem cells

doi: 10.1111/jcmm.12529

Figure Lengend Snippet: Hematopoietic stem cells receive microRNA loaded extracellular vesicles released by Cy5-RNA positive telocytes. (A) Experimental set-up : Cardiac telocytes are transfected with lipofectamine/Cy5-RNA complexes and release extracellular vesicles loaded with fluorescent-RNA into culture supernatant; hematopoietic stem cells incubated with supernatant receive the Cy5-RNA vesicles and become fluorescent; in control experiments, telocytes are not transfected with oligoRNA in absence of lipofectamine, the released vesicles does not have Cy5-RNA and the corresponding hematopoietic stem cells are negative for Cy5 fluorescence. (B) TC transfection with Cy5-RNA : Rat or mouse telocytes are positive for fluorescent Cy5 after 3–5 hrs incubation with a mixture of Cy5-RNA oligo and Lipofectamine RNAiMAX (red filled areas). Controls, non-fluorescent telocytes, represent cells incubated with oligoRNA in absence of lipids (dot plots). (C) Cy5-RNA positive hematopoietic stem/progenitor cells : Hematopoietic progenitors (LSK) are fluorescent after incubation with supernatant derived for Cy5-RNA transfected telocytes (sample), but not in case of supernatant derived from telocytes negative for Cy5 (control). LSK cells Lineage/7AAD negative, Sca1 positive and c-Kit positive. N = 4, P = 0.03 (D) Representative FACS plots of data presented in (C) . Right plot: Hematopoietic stem cells (HSC) and their multipotent progeny (MPP), defined as cells LSK Flt3 negative, are positive for Cy5 when cultured in presence of medium conditioned by Cy5-RNA transfected telocytes. Left plot: in control experiments, HSC/MPP have been incubated with conditioned supernatant derived from non-fluorescent telocytes.

Article Snippet: The immunophenotype of hematopoietic cells was determined by cell staining with the following antibodies: anti-cKit APC-Cy7 (clone 2B8; Biolegend, San Diego, CA, USA), anti-Sca1 PB (clone E13-161.7; Biolegend), anti-CD135 (Flt3) PE (clone A2F10; Biolegend), anti-CD150 Brilliant VioletTM 510 (clone TC15-12F12.2; Biolegend), biotinylated anti lineage panel (# 133307; Biolegend), Streptavidine PE-Cy5 (# 554062; Becton Dickinson and Company).

Techniques: Transfection, Incubation, Control, Fluorescence, Derivative Assay, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Telocytes transfer extracellular vesicles loaded with microRNAs to stem cells

doi: 10.1111/jcmm.12529

Figure Lengend Snippet: Cardiac stem cells receive microRNA loaded extracellular vesicles released by Cy5-RNA positive telocytes. (A) Rat cardiac stem cells are Cy5 positive after incubation with supernatant derived from fluorescent telocytes (right plot). In the control experiment, cardiac stem cells were incubated with supernatant derived from Cy5-RNA free telocytes (left plot). The figure shows representative data of 4 independent experiments. (B) Statistical data of 4 experiments ( T = 0.02).

Article Snippet: The immunophenotype of hematopoietic cells was determined by cell staining with the following antibodies: anti-cKit APC-Cy7 (clone 2B8; Biolegend, San Diego, CA, USA), anti-Sca1 PB (clone E13-161.7; Biolegend), anti-CD135 (Flt3) PE (clone A2F10; Biolegend), anti-CD150 Brilliant VioletTM 510 (clone TC15-12F12.2; Biolegend), biotinylated anti lineage panel (# 133307; Biolegend), Streptavidine PE-Cy5 (# 554062; Becton Dickinson and Company).

Techniques: Incubation, Derivative Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Telocytes transfer extracellular vesicles loaded with microRNAs to stem cells

doi: 10.1111/jcmm.12529

Figure Lengend Snippet: Telocytes receive microRNA loaded extracellular vesicles released by Cy5-RNA positive cardiac stem cells. Cardiac telocytes are Cy5 positive after incubation with culture supernatant derived from fluorescent cardiac stem cells (right plot). In the control experiment, telocytes were incubated with supernatant derived from Cy5-RNA free cardiac stem cells (left plot). The figure shows representative data of 2 experiments.

Article Snippet: The immunophenotype of hematopoietic cells was determined by cell staining with the following antibodies: anti-cKit APC-Cy7 (clone 2B8; Biolegend, San Diego, CA, USA), anti-Sca1 PB (clone E13-161.7; Biolegend), anti-CD135 (Flt3) PE (clone A2F10; Biolegend), anti-CD150 Brilliant VioletTM 510 (clone TC15-12F12.2; Biolegend), biotinylated anti lineage panel (# 133307; Biolegend), Streptavidine PE-Cy5 (# 554062; Becton Dickinson and Company).

Techniques: Incubation, Derivative Assay, Control