EMBO/SourceData huggingface dataset:
Figure 2A. Left panel: comparison of normalized EGF-Alexa647 (160 ng/ml; 5') fluorescence intensity bound to individual endogenous EGFR expressing MCF10A (yellow), to exogenous EGFR-mCitrine expressing EmCit_MCF7 cells (black) and WT MCF7 cells (red); Right panel: normalized EGF-Alexa647 fluorescence plotted against normalized EGFR-mCitrine fluorescence intensity in WT (red) and EmCit_MCF7 (black, with 2nd order polynomal fit: grey line) cells. N=3 biological replicates, n>75 cells, mean±SD.D. Gray: αL upon each administered dose for individual EmCit_MCF7 cells to cumulative doses of EGF-Alexa647 (2.5-640 ng/ml). N=3 biological replicates, n=13 cells. Black: EGF-Alexa647 bound to WT MCF7 cells at the indicated concentrations normalized to the mean fluorescence intensity at 160 ng/ml EGF-Alexa647 (Fig 1E; mean±SD, N=5 biological replicates, n=16-19 fields of view).F. Left: EGFR- (pY1068-) phosphorylation response in WT MCF7 cells obtained from western blots normalized to maximal phosphorylation obtained by inhibiting all phosphatases by 0.33 mM pervanadate (N=6; red symbols with mean±SD and fit to the hill equation (solid line)) at 0 (plotted as 0.001 to fit the logarithmic x-axis), 0.5, 1, 2, 5, 10, 20, 40 and 80 ng/ml plotted against corresponding αL (obtained from in cell dose response experiments in EmCit_MCF7 cells (D)). Correspondig molecular RPTPγ/EGFR-ratio (see G, H, Fig EV2H) is depicted above the graphs; Inserted into each graph are the values of Hill coefficient (HC) and EC50 of the fitted hill equation (95% confidence interval). 2nd graph: Same as left graph with αp plotted vs αL both obtained from in cell dose response experiments in EmCit_MCF7 cells. 3rd-5th graph: Same as 2nd graph for EmCit_MCF7 RPTPγ-KO with RPTPγ-mTFP ectopic expression clustered by RPTPγ/EGFR-expression ratio and HC (Fig EV2G).G. Number of molecules obtained by normalizing background-subtracted fluorescence intensities of individual transfected cells against the mean background intensity of untransfected cells, yielding relative expressions levels independent on the fluorophore (methods). This value was then set into proportion to the known mean number of EGFR per MCF10A cell to yield absolute molecule count/cell. 1st column: EGFR-mCitrine in EmCit_MCF7 cells (N=3 biological replicates, n=102 cells); 2nd column EGFR-mCitrine expressed in EmCit_MCF7 RPTPγ-KO expressing RPTPγ-mTFP (N=3 biological replicates, n=26 cells); 3rd column: RPTPγ-mCitrine in MCF7 cells expressing additionally EGFR-mCherry (N=3 biological replicates, n=253 cells); 4th column: RPTPγ-mTFP expressed in EmCit_MCF7 RPTPγ-KO (N=3 biological replicates, n=26 cells) (individual cells with mean+SD). Color code in 2nd and 4th column is attribution to respective cluster (H and EV2G).
Figure 3C-E (C) in cell EGF-dose response imaging for RPTPγ-mCitrine oxidation. Left panel: Representative confocal micrographs of RPTPγ-mCitrine in EmTFP_MCF7 cells (top row) together with its oxidized fraction estimated using DyTo-FLIM (αox, bottom row), upon 10' stimulation with EGF-Alexa647 (0-160 ng/ml) including 5' together with 0.5 mM DyTo. Scale bar: 10 μm. Right panel: Quantification depicting the PM-proximal (orange) and PM-distal (blue) oxidized fractions as functions of receptor occupancy (αL) and corresponding EGF-Alexa647, or H2O2 concentration in EmTFP MCF7 cells expressing RPTPγ-mCitrine (WT) or RPTPγC1060S-mCitrine (C1060S) as well as WT cells treated with 0.5 mM atto590 instead of DyTo (atto590). Mean of individual cells (symbols) with mean±SD (black lines), N=3 biological replicates, n=13-15 cells per EGF dose. P: unpaired two-tailed t-test, between PM (serpentine peripheral structures) and endosomal (vesicular structures) fractions. (D) Same as in (C), for RPTPγ-mCitrine oxidation in p22phox-KO cells. N=3 biological replicates, n=14-26 cells per EGF dose. (E) Same as in (C), for TCPTP-mCitrine or TCPTPC216S-mCitrine (C216S) oxidation in EmTFP_MCF7 cells. N=3 biological replicates, n=18-21 cells per EGF dose.
Figure 4A. Representative confocal micrographs of MCF7 WT cells showing the co-localization of RPTPγ-mCitrine (1st column: green; 3rd column: blue) and EGFR-mCherry (2nd column: green; 3rd column: yellow) with recycling-endosome defined by immunostaining against Rab11a (magenta), without (top row) or after 30' EGF-DyLight405 stimulus (160 ng/ml; bottom row). Scale bar: 10 μm.B. Fraction of RPTPγ-mCitrine (cyan) or EGFR-mCherry (green) that spatially overlaps with Rab11a (top left, N=3, n=23-26 cells per timepoint), PM (bottom left, N=3 biological replicates, n=15-17 cells), EEA1-positive early endosomes (top right, N=3 biological replicates, n=25-28 cells) or Rab7-positive late endosomes (bottom right, N=3 biological replicates, n=23-27 cells) in MCF7 cells as function of time after 160 ng/ml EGF-stimulus. Orange symbols/dotted line: same for EGFR-mCitrine in RPTPγ-KO cells (N=3 biological replicates, n=17-21 cells). P: unpaired two-tailed t-test; colored P values compare to the respective species before stimulation, black in between species.C. Left: Representative confocal micrographs of MCF7 cells depicting the steady state localization of expressed RPTPγ-mCitrine (cyan), without (top) or with co-expression of BFP-Rab11a (yellow, bottom). Top right: Quantification of PM-localized fraction of RPTPγ-mCitrine without (WT) and with co-expression of BFP-Rab11a. Bottom right: Fraction of RPTPγ-mCitrine localized to the PM in individual cells as a function of BFP-Rab11a expression level, measured by mean BFP-fluorescence intensity. Scale bar: 10 μm. N=2 biological replicates, n>40 per condition, mean±SD,; P: unpaired two-tailed t-test.E. Upper panel: Dual-color widefield images (1st column), SRRF reconstructions (2nd column) with magnifications of boxed areas (3rd column) of Alexa647-SNAP-EGFR (green) and RPTPγ-mCitrine (magenta) of cryo-arrested MCF7 cells, unstimulated (top row) or stimulated with 100 ng/ml EGF (bottom row) for 15'. Scale bar: 10 μm. Lower panel: corresponding Manders colocalization coefficients for Alexa647-Snap-EGFR/RPTPγ-mCitrine from SRRF reconstructions on intracellular compartments or PM area for unstimulated (n=12-18) and 15' EGF-stimulated (n=13-14) cells. mean±SD, P: unpaired two-tailed t-test.F. Left: Representative IP-western blot showing co-IP of EGFR (2nd row) upon RPTPγ-mCitrine (1st row: lanes 1-6) or RPTPγC1060S-mCitrine (lane 7) pull-down by anti-GFP antibody from lysates of MCF7 cells co-transfected with EGFR and RPTPγ-mCitrine or RPTPγC1060S-mCitrine: without stimulus (0 ng/ml), upon 10' stimulus with EGF-Alexa647 (5-320 ng/ml, also displayed as corresponding receptor-occupancy αL, Fig 2D) or 8 mM of H2O2. 3rd and 4th row: total protein concentrations of RPTPγ-mCitrine and EGFR in the lysate measured by western blot as input control for the Co-IP. Right: corresponding ratiometric quantification of co-immunoprecipitated EGFR over pulled down RPTPg-mCitrine or RPTPγC1060S-mCitrine protein bands (mean±SD, N=4 biological replicates, P: unpaired two-tailed t-test)
Figure 5. C. Catalytic (ε1 − ε4) and autocatalytic (α1 − α4) rate constants obtained from iterative global fitting the ODEs in (B) solved for steady state (dEGFR p/T/dt = dEGF-EGFRp/T/dt = dRPTPγA/T/dt = 0) to EGF-dose response (aL − aP) data from all EGFR and PTP expression conditions (Fig 2E, F, I). EGFRp, EGF-EGFRp: product of the corresponding reaction. F. Change of the free parameter groups Γ1 = γ1RPTPγ/EGFRT and Β = β EGFRT/k1 in EmCit_MCF7 RPTPγ-KO (blue), EmCit_MCF7 (red), EmCit_MCF7 RPTPγ-KO expressing RPTPγ-mTFP splitted in 3 clusters with increasing RPTPγ−mTFP/EGFR-mCitrine ratio (yellow, green, purple; Fig 2H, EV2G) and WT MCF7 cells (black
Figure 6C. Top: Representative Western blot against Erk and phosphorylated Erk (pT202 and pY204) in WT (red) compared to p22phox-KO (green) MCF7 cells after the indicated times of sustained stimulation with 20 ng/ml EGF-Alexa647. Bottom: Corresponding quantification of the fraction of phosphorylated ERK as a function of stimulation time. Mean±SD, N=4 biological replicates, P: unpaired two-tailed t-test.D. Quantification of cell proliferation using retinoblastoma (Rb) protein phosphorylation detected by immunofluorescence, for WT (red), RPTPγ-KO (blue) and p22phox-KO (green) MCF7 cells without or post 24 h of EGF-Alexa647 treatment (1, 20, 160 ng/ml). Mean±SEM, N=3 biological replicates, n>2000 cells per EGF stimulus per cell line, P: two-way ANOVA with Tukey multiple comparisons.E. Quantification of the culture-well area (%) occupied by proliferating cell-colonies, obtained from clonogenic assays of WT, RPTPγ-KO and p22phox-KO MCF7 cells, plated either in medium containing 20 ng/ml EGF and 0.5% FCS (left: orange bars, N=3-4 biological replicates, 11-12 wells each) or complete serum growth medium containing 10% FCS (right: pink bars, N=4 biological replicates, 12 wells each). Mean±SEM, P: unpaired two-tailed t-test with Welch's correction.
Figure 1(B) Representative immunoblot analysis of CALHM6 expression in BMDM and BMDC stimulated for 6 or 24 h with Poly(I:C) (50 μg/ml) or LPS (100 ng/ml) (one representative experiment of two is shown, n = 2 mice).(G and H) Calhm6 mRNA expression measured by qPCR in naïve or IFN-γ (10 ng/ml) primed (6 h) BMDM stimulated with (G) Poly(I:C) (50 μg/ml) (one representative experiment of two is shown, n = 2 mice, biological replicates), or (H) Zymosan (100 μg/ml) for 2 to 24h (one representative experiment is shown, n = 3 mice, biological replicates).
Figure 2CFU/g of spleen are shown (B) after 24 h (pooled results from two independent experiments, WT mice = 9, Calhm6-/- mice = 12, Man Whitney test), (C) 72 h (pooled results from two independent experiments, WT mice = 16, Calhm6-/- mice = 17, Man Whitney test)Serum collected from infected mice at peak infection (day 3) and serum cytokines quantified using LEGENDplex Mouse Inflammation Panel assay to determine IFN-γ (F), IL-6 (G) and IL-10 (H) concentration (pooled results from two independent experiments, WT mice = 16, Calhm6-/- mice = 17, Mann Whitney test).
Figure 3(F-J) WT and Calhm6-/- mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS for 3 h (F-H) or 12 h (I-J), spleens were collected and processed as in (A-E). Representative contour plots (F, I), pooled frequency of IFN-γ+ in CD3-NK1.1+ cells of WT vs Calhm6-/- mice (G,J) and ratio of IFN-γ+ cells that are NK1.1+ or CD3+ normalised as in C (H) (For Poly(I:C) 3 h: pooled results from two independent experiments, Poly(I:C) WT mice = 10, Calhm6-/- mice = 10, control WT mice = 2, Calhm6-/- mice = 2, One way ANOVA with multiple comparisons. For Poly(I:C) 12 h: results from one experiment, Poly(I:C) WT mice = 6, Calhm6-/- mice = 5, control WT mice = 1, Calhm6-/- mice = 1 mice, One-way ANOVA with multiple comparisons).
Figure 4(A-B) WT and Calhm6-/- mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS. On day 3 spleens were collected, stained with antibodies against NK cell-maturation markers and analysed by flow cytometry. Representative contour plots (A) and pooled flow cytometry results (B) are shown (results from one experiment, Poly(I:C) WT mice = 5, Calhm6-/- mice = 5, control WT mice = 2, Calhm6-/- mice = 2 mice, One-way ANOVA with multiple comparisons).(F) NO production in WT and Calhm6-/- BMDM treated overnight with LPS or Poly(I:C) quantified with a Griess test on cell supernatant (results from one experiment, WT mice = 3, Calhm6-/- mice = 3, Kruskal Wallis test with multiple comparisons).(G) BMDM with or without IFN−γ priming were grown overnight in antibiotic free medium. Cells were infected with L. monocytogenes (MOI 1) for 30 min. Gentamicin (5 ng/ml) was then added to the medium to kill extracellular bacteria. At 0, 4 and 8 h cells were harvested, lysed with 0.1% Triton X-100 and the lysate was plated on antibiotic free BHI plates for 24 h, and CFU were quantified (pooled results from three independent experiments, WT mice = 3, Calhm6-/- mice = 3, Two-way ANOVA test).
Figure 5(D-G) BMDM were transduced with a retroviral vector expressing CALHM6-eGFP. BMDM (3x105) were plated on coverslips and pre-treated with IFN-γ and LPS for 6 h. (D) Freshly isolated primary CD3-NK1.1+ cells isolated from mouse spleens were added for 0, 10, 20, or 40 min on top of plated BMDMs that were generated from bone marrow cells transduced with a retroviral vector expressing CALHM6-eGFP. The interacting cells were then fixed and stained with phalloidin to identify actin and DAPI to identify nucleus. Synaptic interaction between BMDM and NK cell is indicated by white arrowheads; entire collapsed imaging Z-stack is shown. (E) Images were blinded and CALHM6-eGFP polarisation towards CD3-NK1.1+ cells manually scored only in Z-slices where BMDM-NK interaction was identified (p-value calculated by two-way ANOVA of polarisation and time points). Numbers on top of each bar indicate number of synapses scored for that time point. (F) IgG2a-opsonised sRBC labelled with PHK26 were added on top of plated CALHM-eGFP-expressing BMDM. One single Z-slice containing BMDM-sRBC interaction plane is shown. Synaptic interaction between BMDM and sRBC cell is indicated by white arrowheads (G) Quantified results of experiment in F. Numbers on top of each bar indicate number of synapses scored for that time point. Longer time points were not possible to score for opsonised sRBC because of the speed of the phagocytosis as sRBC were quickly internalised. Images taken with 60x objective (p-value calculated by two-way ANOVA of polarisation and time points).
Figure 6(B) WT or Calhm6-/- BMDM stimulated with IFN-γ (10 ng/ml) for 24 h were lysed with 1% Triton X100-containing buffer with cOmplete protease inhibitor cocktail and treated with increasing concentrations of DTT. The samples, without boiling, were resolved by SDS-PAGE gel (one representative experiment of two is shown, n = 4 mice). Lack of DTT influences solubilisation of GADPH monomer, and its detection for immunoblotting.(G) Current-voltage (I-V) relations for WT-mCALHM6, E119R-mCALHM6 and control (ASO) in 2-mM Ba2+ bath solution (pooled individual whole-cell patches, WT-mCALHM6 n = 13, E119R-mCALHM6 n = 4, control = 4, SEM error bars).
Figure 7(A) Fura-2 fluorescence ratio (F340/380) recorded in N2a cells expressing human CALHM1 (hCALHM1), mouse CALHM6 (mCALHM6) or control (vector) in response to removal and subsequent add-back of extracellular Ca2+. Traces representative of four independent experiments for each, normalized to baseline ratio. Right: Resting F340/380 ratio in N2a cells expressing hCALHM1, mCALHM6 or vector in 2 mM Ca2+ (the error bars represent SEM).(E) Steady-state ATP release from activated macrophages: BMDMs were plated at 150,000 cells/well in a 48-well plate, and incubated for 3 h with Poly(I:C) (50 μg/ml), LPS (100 ng/ml) and ATPase inhibitor ARL67156 (1 mM) in culture medium. Control cells were incubated with 1 mM ARL67156 only. ATP release (nM) measured using Sigma ATP Bioluminescent Assay: WT without stimulation: 9.12 ± 0.76; WT with stimulation: 22.16 ± 1.83; Calhm6-/- cells without stimulation: 8.76 ± 0.55; KO cells with stimulation: 12.19 ± .75. (pooled results from three independent experiments, WT mice = 3, Calhm6-/- mice = 3, Two-tailed unpaired Student's T test, error bars represent SEM).
Figure 1C, Survival assays against substrates of QacA showed loss of activity against monovalent cationic antibacterials (TPP: Tetraphenylphosphonium, Et: Ethidium), while still retaining partial activity against divalent cations (Dq: Dequalinium, Ch:Chlorhexidine). EV: pBAD empty vector. n = 3 for biological replicates. CFU indicates colony forming units. Image color was inverted from black to white background to enhance clarity.E, Population shift-based titration of QacA-GFP against ICabs yielded an apparent Kd of 65 ± 4 nM for QacA-A4 complex and 100 ± 10 nM for QacA-B7. n = 3 for independent replicates. Error bars denote S.E.M. and each datapoint represents mean value.G, Colocalization of QacAWT or QacAD411N (tagged with GFP) on E. coli spheroplasts (stained with DAPI for viability) with ICabs (labelled with NHS-Rhodamine) was screened against ICab A4 (left) and ICab B7 (right). n = 10-30 spheroplasts were imaged for each sample.H, Representative reference-free 2D classes of QacAD411N embedded in UDM micelle, in complex with ICab A4 (top) and with ICabs A4 and B7 (bottom), pointed with pink and green arrows respectively. Representative classes shown for QacA-A4 complex are from a set of 68,000 particles and QacA-A4/B7 complex are from 218,040 particles.
Figure 2A, CryoEM model of QacA (blue) in complex with ICab A4 (pink) and ICab B7 (green)
Figure 4C, Whole cell ethidium efflux assay with overexpressed EL7 deletion construct in the background of wild type and D411N mutant. n = 3 for independent replicates, one of which is shown here. Error bars represent S.E.M. from six technical replicates.D, Survival assays of QacAΔEL7 and QacAD411N/ΔEL7 mutants in the presence of 10 μM Tetraphenylphosphonium (TPP) and 16 μM Chlorhexidine (Ch). Data from one representative biological replicate is shown here. Image colors were inverted for improved clarity.
Figure 5C Whole cell ethidium efflux assay done with E.coli JD838 cells expressing QacAWT or QacA5A. Empty vector (EV) was used as a control for the experiment. A graph of one independent replicate is shown with error bars representing S.E.M. of 6 technical replicates.D A single representative replicate of survival assays shown for the same cells spotted in 10-fold dilution series on 12 μM TPP and 16 μM Ch. Image colors were inverted for clarity in the figure.G Survival assays of solely EL7 chimeric constructs compared against empty vector (EV) and QacAWT. One representative biological replicate is shown. Image color inverted for clarity.Whole cell-based ethidium efflux assay done with E.coli JD838 cells expressing QacAWT, its EL7 deletion construct (QacAΔEL7), and the chimeric mutants of LfrA and SmvA. All assays were done in at least 2 independent replicates. One representative replicate shown with data normalized to highest value in each trace. Error bars depict S.E.M. for six technical replicates.I Whole cell-based ethidium efflux assay done with E.coli JD838 cells expressing QacAWT, its EL7 deletion construct (QacAΔEL7), and the chimeric mutants of LfrA and SmvA. All assays were done in at least 2 independent replicates. One representative replicate shown with data normalized to highest value in each trace. Error bars depict S.E.M. for six technical replicates.J Everted vesicles-based assays of chimeric and EL7 deletion constructs of QacA. Traces represent mean values of three replicates performed on vesicles made with a single batch of cells for each construct. Timepoints indicated as 1-3 above the traces indicate addition of 100 μM ATP, 1 mM TPP and 2 μM Nigericin respectively. One representative shown for data obtained from biological triplicates. Error bars represent SEM for technical triplicates from one of three biological replicates.
Figure 1F-M) Total cellularity of F) HSPCs, G) LT-HSCs, H) ST-HSCs, I) Tom+ HSCs, J) GFP+ drHSCs, K) MPP2, L) MPP3, M) MPP4 in E16.5 fetuses following saline or maternal infection with Pru or RH as shown in Fig. 1A. n = 9-15 fetuses from at least 3 litters/condition.N-O) Frequency of N) HSCs (CD150hi) O) MPPs (CD150- HSPCs) expressing Ki67 (G1+G2-M-S) at E16.5 following saline or maternal infection with Pru or RH n = 9-15 fetuses from at least 3 litters/condition. For all analysis above bars represent mean + SEM. One-way ANOVA with Tukey's test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
Figure 2B) Ratio of primary recipients with long-term multi-lineage reconstitution (LTMR: > 1% peripheral blood chimerism within each mature lineage) in all 4 lineages (granulocyte/macrophages [GM], platelets, B- and T-cells), n is shown as the numerator. Statistical significance determined by Fisher's exact test (2-tailed). *p ≤ 0.05C-F) Peripheral blood (PB) chimerism of primary Tom+ HSC transplant recipient mice with LTMR in C) granulocytes/macrophages (GM), D) platelets, E) B-cells, and F) T-cells at week 16 post-transplantation.G-J) Peripheral blood (PB) chimerism of primary GFP+ drHSC transplant recipient mice with LTMR in G) granulocytes/macrophages (GM), H) platelets, I) B-cells, and J) T-cells at week 16 post-transplantation.K) Ratio of secondary recipients with long-term multi-lineage reconstitution (LTMR; > 0.1% PB chimerism) in all 4 lineages (GM, platelets, B- and T-cells), n is shown as the numerator. Statistical significance determined by Fisher's exact test (2-tailed). *p ≤ 0.05L-O) Peripheral blood (PB) chimerism of secondary recipients of Tom+ HSC with LTMR in L) granulocytes/macrophages (GM), M) platelets, N) B-cells, and O) T-cells at week 16 post-transplantation.P-S) Peripheral blood (PB) chimerism of secondary recipients of GFP+ drHSCs with LTMR in P) granulocytes/macrophages (GM), Q) Platelets, R) B-cells, and S) T-cells at week 16 post-transplantation. No reconstitution (nr) was present in recipients of GFP+ drHSCs in RH. Graphs C-J and L-S are plotted as the mean + SD. One-way ANOVA with Tukey's test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
Figure 3A-I Measurement of A) IFNγ, B) IFNβ, C) IL-1β, D) IL-10, E) IL-1α, F) TNFα, G) IL-6, H) IL-27, and I) GM-CSF cytokines in fetal amniotic fluid at E15.5 following prenatal infection. n=8-9 fetuses from at least 3 litters/condition. For all analysis above bars represent mean + SD. One-way ANOVA with Tukey's test. *p ≤ 0.05; **p ≤ 0.01; **p≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.U-C') Measurement of U) IFNγ, V) IFNβ, W) IL-1β, X) IL-10, Y) IL-1α, Z) TNFα, A') IL-6 B') IL-27, and C') GM-CSF cytokines in maternal serum at E15.5 following prenatal infection; n=3 mice/condition. For all analysis above bars represent mean + SD. One-way ANOVA with Tukey's test. *p ≤ 0.05; **p ≤ 0.01; **p≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
Figure 4A-H) Total cellularity of A) HSPCs, B) LT-HSCs, C) Tom+ HSCs, D) GFP+ drHSCs, E) ST-HSCs, F) MPP2, G) MPP3, and H) MPP4 at E15.5 following maternal IFNγ injection. For all analysis above, bars represent mean + SEM. n = 9-13 fetuses from 3-4 litters/condition. Statistical significance was determined by unpaired student's t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
Figure 5A) Ratio of recipients with long-term multi-lineage reconstitution (LTMR: > 1% PB chimerism) in all 4 lineages (GM, platelets, B- and T-cells). N of mice is shown as the numerator.B-E) Peripheral blood (PB) chimerism of B) granulocyte/macrophages (GM), C) platelets, D) B-cells, and E) T-cells in Tom+ HSC transplant recipients at 16 weeks post-transplantation. F-I) Peripheral blood (PB) chimerism of F) granulocyte/macrophages (GM), G) platelets, H) B-cells, and I) T-cells in GFP+ drHSC transplant recipients of mice at 16 weeks post-transplantation.J) Ratio of secondary transplant recipients with long-term multi-lineage reconstitution (LTMR > 0.1% PB chimerism) in all 4 lineages (GM, platelets, B- and T-cells). N of mice is shown as the numerator.K-N) Peripheral blood (PB) chimerism of K) granulocyte/macrophages (GM), L) platelets, M) B-cells, and N) T-cells in secondary Tom+ HSC transplant recipients at 16 weeks post-transplantation. O-R) Peripheral blood (PB) chimerism of Q) granulocyte/macrophages (GM), R) Platelets, S) B-cells, and T) T-cells in secondary transplant recipients of GFP+ drHSCs at 16 weeks post-transplantation. For all % chimerism experiments, mice not demonstrating LTMR were not included in analysis. Bars represent mean + SD. Statistical significance was determined by unpaired student's t-test. *p ≤ 0.05; **p ≤ 0.01.