EMBO/SourceData huggingface dataset:
Figure 2(A - C) Adult C57BL/6J mice (four months old) were intraperitoneally injected with DFO at 100 mg/kg three times a week for three months in a row. SW-AF images were acquired longitudinally at one, two and three months post injection.(I, J) Flat mount RPE was harvested from the mice treated with DFO for five months and stained by TUNEL to detect cell death. Hoechst was used for nuclear staining. Untreated mice of similar age were used as the healthy controls. Flat mount images were captured by 10× confocal microscopy and stitched together.(K - N) The RPE was dissected from the mice injected with DFO for three and nine months respectively and stained with phalloidin. Age-matched untreated mice were included as the controls. The images were taken at 40× magnification.
Figure 3(A - D) Light microscopic examination of the iRPE cells 24 and 48 hours post treatment. Scale bar: 50 µm.(E - H) iRPE cells were stained with ZO-1 24 and 48 hours post treatment. Untreated iRPE cells were used as the control.(I - L) Cell death among the iRPE population was examined by TUNEL assay 24 hours and 48 hours post treatment. Untreated iRPE cells were included as the control. The cells were counterstained with Hoechst.
Figure 4(A) iRPE cells treated with DFO were collected and lysed to measure the expression of HIF1α and HIF2α 24 and 48 hours after the treatment. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the control. Actin was used as the loading control. HIF1α (post-translationally modified) was predicted to be ~100 kDa; HIF2α (post translationally modified) was predicted to be ~ 120 kDa. (B) iRPE cells treated with DFO were lysed and fractionated by centrifugation to measure the expression of HIF1α and HIF2α in the nuclei 24 and 48 hours after the treatment. Untreated iRPE cells were used as the normal control. Histone H3 was used as the loading control. (
Figure 5(A) Ferrous iron was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05.(B) The transcripts of ferroptosis-associated markers were determined by qPCR using iRPE cells as the template. The RNA extract from untreated iRPE was included as the control. The level of each transcript was normalized to ACTB. The statistics are analyzed by ratio paired student's t-test for each target gene. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05; ** p < 0.01. Round dots: untreated iRPE; square dots: DFO-treated iRPE.(C) The level of ROS was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 6 iRPE lines for each group. * p < 0.05, ** p < 0.01. DHE: Dihydroethidium.(D) Mitochondrial respiration of the iRPE cells with DFO treatment was determined by seahorse extracellular flux assay. OCR was determined over a course of time. Each data point is shown as mean ± S.E.M., n = 4 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.
Figure 6(A - C) Light microscopy of morphological protection of AKG against DFO toxicity on iRPE. The cells were treated with DFO at 100 mM for 48 hours (B). AKG was supplemented at 10 mM simultaneously (C). The control group was supplemented with 0.1% DMSO (A). Scale bar: 100 µm. (D - F) The DFO-treated iRPE cells with or without AKG supplementation were subject to phalloidin staining (E and F). The iRPE cells treated with 0.1% DMSO-containing media were included as the controls (D). The images were captured at 40× magnification. Scale bar: 30 µm. (G - I) Detection of cell death in the DFO-treated iRPE cells with or without AKG supplementation by TUNEL staining. Hoechst was used for nuclear staining. The images were captured at 40× magnification. Scale bar: 30 µm. (J(J - L) SW-AF was performed longitudinally on the mice treated with DFO for up to 10 months in conjunction with supplementation of AKG for seven months.
Figure 7(A) Mitochondrial respiration of the DFO-treated (100 mM) iRPE cells with AKG supplementation (10 mM) for 24 hours was determined by seahorse extracellular flux assay. OCR was measured over a course of time. Each dot is shown as mean ± S.E.M., n = 5 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.(G) The DFO-treated (100 mM) iRPE cells supplemented with AKG (10 mM) for 48 hours were lysed for immunoblotting against HIF2α. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the controls. HIF2α (post translationally modified) was predicted to be ~ 120 kDa. Actin was included as the loading control. (H) RPE lysate from the one-year-old mice subject to 10-month DFO treatment with or without concomitant supplementation of AKG was used for immunoblotting against HIF2α. The RPE of the age-matched untreated mice was included as the control. HIF2α (degraded) was predicted to be 40 - 80 kDa. Actin was used as the loading control.
Figure 1Macrophages produce little to no inflammatory cytokines when incubated with either (a) whole iRBC for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBCMacrophages produce little to no inflammatory cytokines when incubated with either (b) fresh iRBCL for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (b) n=5 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC(c) Macrophages were pre-incubated with IFNγ (1000U/ml) for 18 h before iRBCL stimulation. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=3 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC(d) Phase-contrast microscopy of macrophages 24 h after the addition of RBC, iRBC or LPS. Dark hemozoin crystals can be observed within macrophages. Bar is 30 µm.(e) Macrophages were incubated with iRBCL 24 h before addition of LPS. Paired t-test was performed to determine statistical significance. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (e) n=3. Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared RBCL (e).
Figure 2Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. RBCL, iRBCL, XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL (a) n=7 One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO, Hypoxanthine (Hyx) were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. XO added to macrophages for 24 h. Febuxostat (Feb) was pre-incubated for 30 minutes with XO before addition to macrophages. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO the anti-oxidants N-acetyl-L-cysteine (NAC) and 1-Thioglycerol (1-TG), and heat inactivated (HI) XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).Cytokine secretion of PBMC (e) incubated with the indicated stimuli for 24 h. RBC, iRBC, RBCL, iRBCL, XO Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).
Figure 3(a) XO was incubated with macrophages for the indicated times, when wells were washed twice and media was replaced. Supernatants were collected for cytokine determinations 24 h later (n=3). Kruskal-Wallis statistical method was used for statistical analysis. Blue asterisks indicate significance when values were compared with time 0 (*p<0.05).(b) Diagram shows experimental set up with macrophages in the bottom of the well and XO added either in the dialysis cassette (DC, blue) or the well (black). XO activity panels show that the enzyme did not leak through the DC membrane. Secretion of IL6 and TNF was measured 24 h after addition of XO in each compartment. A representative result of two independent experiments is shown.
Figure 4(a) Plasma levels of TNF and IL8 from patients with uncomplicated (black circles, n=14) or cerebral (red circles, n=9) malaria correlate with levels of XO-produced ROS detected in each sample. ROS is expressed as fold change over plasma from healthy controls. Uncomplicated malaria, gray circles, cerebral malaria, red circles. Linear regression (a) were performed to determine statistical significance (*p<0.05, **p<0.01).(b) Levels of XO activity in malaria patients correlate with disease severity UM (uncomplicated malaria), CM (cerebral malaria). Mann-Whitney test (b) were performed to determine statistical significance (*p<0.05, **p<0.01).(c) Plasma from malaria patients induce macrophages to secrete cytokines, which are inhibited by a XO specific inhibitor. Plasma from a healthy control (HP) and from three patients: P1 (with cerebral malaria), P2 and P3 (both with uncomplicated malaria) was pre-incubated for 30 min with febuxostat (Feb) or alone at 37ºC before addition to macrophages at 1:2 dilution in media for 30 min. Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation. unpaired t-tests (c) were performed to determine statistical significance (*p<0.05, **p<0.01).
Figure 5Cytokine secretion by macrophages after 24 h incubation with the indicated stimuli. (b) IL1β levels after addition of increasing concentration of RBCL or iRBCL (1:2, 1:4 and 1:8 macrophage:iRBCL). One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; (b) n=2 Insets show paired samples by donor in two different conditions.chemokine secretion by macrophages after 24 h incubation with the indicated stimuli. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=4. Insets show paired samples by donor in two different conditions.
Figure 6(a) Gene expression analysis of macrophages incubated with the indicated stimuli expressed as fold change over RBCL. Dotted red line indicates 2-fold increase in RNA expression.(b) Western Blot analysis of proIL1β. Relative abundance is the average of 3 independent experiments with different donors.(c) FLICA analysis of activated caspase-1. Results are representative of 2 independent experiments with different donors.(d) Two distinct siRNAs against NLRP3 and an irrelevant control siRNA were utilized to knock down RNA levels by lipofectamine transfection in macrophages that were later incubated with iRBCL, or RBCL as control, with or without XO, before quantification of secreted IL1β. Results are representative of 2 independent experiments with different donors.
Figure 1(B, C) Levels of anti-HEL IgG in serum as measured by ELISA at the indicated days post immunization with HEL-OVA/CFA following adoptive transfer of BHEL cells without CD4OT-II cells (no T cell control) or with control CD4OT-II cells or NEF-expressing CD4OT-II cells. (B) NEF WT of HIV-1SF2 (C) patient-derived NEF variants. Shown are relative levels of HEL IgG production with the mean of IgG levels in mice receiving control cells between days 12 - 14 post immunization arbitrarily set to 1. Each dot represents one animal. Bars represent mean values with SD from 2-10 mice analyzed from 3 (B) and 2 (C) independent experiments. Statistical significance was assessed by Mann Whitney U test. ***P ≤0.001, *P ≤0.05
Figure 2(E) Representative confocal micrographs of p-tyrosine (p-Tyr) polarization at B-T cell contacts after 1-2h of co-culture. Cell conjugates were stained with anti-pTyr antibody. Scale bar, 5µm. The top panels display only the p-Tyr signal in grey, the bottom panel depicts p-Tyr (red), BHEL cells (blue) and CD4OT-II cells (green). (F) Quantification of p-Tyr accumulation at the B-T interface as shown in E. Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment. (G) NEF prevents induction of F-actin remodeling in BHEL during IS. BHEL cells (blue) and CD4OT-II cells (green) were placed on PLL-coated slides and subjected to live-cell time-lapse imaging. Shown are representative still images from the indicated time points post contact formation (see Movies EV1 and EV2). Red signals indicate actin polymerization specifically in BHEL cells. Scale bars, 5µm. Filled and empty arrowheads indicate examples of accumulation of polarized actin or p-Tyr signal at B-T interface during IS formation or lack thereof, respectively. Asterisks mark pairs of interacting T and B cell. (H) Quantification of B cell actin polymerization as shown in G with the frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment. (L) Relative B cell proliferation (quantified by gating on the last 3-4 generations) is plotted as mean values with SD from four independent experiments. Control T cells +Ag were arbitrarily set to 1.
Figure 3(B) Production of anti-HEL IgG at the indicated day post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and CD4OT-II cells expressing NEF or various NEF mutants. Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post immunization arbitrarily set to 1. Each dot represents one animal. Shown are mean values with SD from 5-12 mice from 4 experiments.(D) Quantification of B cell actin polymerization as shown in C with frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition.(H) Relative surface expression of CD4, CD28, OX40, ICOS, CD45 and CD54 on T cells expressing NEF WT or the indicated NEF mutants. Analysis of surface expression was performed using flow cytometry 3-7 d post transduction of T cells. Depicted are mean values with SD from 3-6 independent experiments, with control T cells arbitrarily set to 100% as indicated by the dashed line.(I) Representative confocal images of the subcellular distribution of IL-4 in CD4OT-II cells expressing the indicated proteins engaged in an IS with BHEL cells. B cells and T cells were placed on PLL-coated cover glasses for 60-90min, fixed and stained for intracellular IL-4. Filled and empty arrowheads indicate presence or absence of IL-4 accumulation at the IS, respectively. Scale bars, 5µm. (J) Quantification of IL-4 accumulation at the IS as depicted in I. Shown are mean values with SD from 3 independent experiments relative to control cells set to 100%.
Figure 4(A) Representative cross-sections of draining LNs (dLNs) from mice 7d post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and transduced CD4OT-II cells. dLN were cryosectioned and stained with fluorophore-conjugated antibodies against GL-7 to reveal GCs (green), CD21/35 to stain for follicular dendritic cells (FDC, red) and Hoechst to stain for cell nuclei (blue). Scale bar, 250µm. (B, C) Quantification of the number of GCs relative to B cell follicles (B) and size of GC (C) on confocal micrographs as shown in A. Shown are mean values with SD from dLNs from three mice in two independent experiment. Each dot represents one LN (3 LNs/mouse) in B and one GC in C.
Figure 6(B) Virus replication as assessed by quantification of RT activity in the cell culture supernatant. Shown are mean values with SD from 6 donors.(C) Production of IgG antibodies against TT from infected blocks at indicated time point. Each dot represents one donor, bar indicates mean value. Tonsillar blocks were infected with indicated virus for 24h. After washing off the virus, blocks were stimulated with TT antigen for 24h. Samples were collected at different time intervals to estimate viral replication and antibody production.
Figure 1Distribution of RT shifts calculated with the -ΔL+ΔE method described in Appendix Figure S1. Different transgenes in a mid-late-replicating region are compared: the IL2R reporter gene under the control of the βA-globin promoter (βA pro) containing an inactive (i, N=10) or an active origin (ii, N=8) flanked by two copies of FIV( 2xFIV), the blasticidin resistance gene (BsR) under the control of the β-actin promoter (β-act pro) (iii, N=7) or a combination of these two transgenes (iv, N=8). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (ns, not significant; **p<0.01; ***p<0.001).
Figure 2A. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:71,000,000-74,100,000 bp, 3.1 Mb; galGal5). RT weighted average (WA) values for the wt and the βA-globin+β-actin cell lines are shown. Early-replicated regions (E) are represented in orange and late replicating regions (L) in blue. Annotated genes (Ref Seq genes) are represented below. The mid-late insertion site is indicated with a red arrow and dotted line.B. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:72,450,000-72,650,000 bp; 200 kb; galGal5). Tracks of nascent strands (NS) enrichments in the four S phase fractions were represented separately (S1 to S4) for the wt and the 2x(βA-globin+β-actin) cell lines. NS enriched and depleted regions for each fraction are represented in purple and blue respectively. Single reads form SNS aligned and track of replication origins (Ori peaks) determined in (Massip et al, 2019) are reported in between. The three mid-late insertion sites 1, 2 and 3 are indicated with red arrows. Annotated genes and CpG Islands are shown below. Initiation zones (IZ) and termination zones (TZ) are reported.
Figure 3A. Distribution of -ΔL+ΔE values for clonal cell lines containing one advanced replicon (βA-globin+β-actin) inserted at site 1 (i, N=8) or 3 (iii, N=3) or at both sites on the same chromosome with one GFP reporter construct composed of the GFP reporter gene under the control of the βA-globin promoter/origin linked to a 1.6 kb fragment of human chromosome 7 (h.K7) inserted at site 2, either on the same chromosome as 1 and 3 (1+2+3), or on the other chromosome (1+3) (ii, N=5). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).C. Interaction profiles of allele 1+3/2 with chr1 for combined Hi-C libraries: distance-corrected interactions, i.e. observed/expected (OE), binned at 500 kb with corresponding compartment ( EV1) tracks. Red arrows represent the loci that show preferred interactions with allele 1+3 and that coincide with A compartment.D. OE interactions of allele 1+3 (orange rectangles) and allele 2 (blue rectangles) averaged over A and B compartments for p and q-arms at 100kb bin size. A random compartment status permutation test was made and then we calculated the one-sided p-value for the observed value of 'difference allele 1+3 / 2 A - B'. Values corresponding to OE interactions of allele 1+3 and allele 2 averaged over shuffled A (red circles, N=1000) and B compartments (blue circles, N=1000) for p and q-arms. ns, not significant; **p<0.01;***p<0.001.E. Scatterplot of density of interactions between allele 1+3 and allele 2 with chromosomes larger than 5,000,000 bp.
Figure 4A. Distribution of -ΔL+ΔE values for clonal cell lines containing one β-actin construct at site 1 (i, N=7) or at site 1 and 3 (ii, N=5), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).B. Distribution of -ΔL+ΔE values for clonal cell lines containing one βA-globin construct at site 1 (i, N=8) or at site 1 and 3 (ii, N=6), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).