EMBO/SourceData huggingface dataset:
Figure 6(A) Comparison of IFNγ production (y-axis) after incubation with plate-coated antibodies or proteins (10 µg/ml each) (x-axis). Box plots showing IFNγ concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition). Data information: Wilcoxon signed-rank test. (A, Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.(B) Comparison of IFNγ levels in the supernatant after co-culture of NK cells with 721.221 target cells in the presence or absence of either 10 µg/ml αTRAIL or isotype control (Iso) using ELISA (n = 8 different donors per condition). Effector:target ratio was 1:1. Left panel: Box plots showing IFNγ levels (y-axis) for the described culture conditions (x-axis). Right panel: Box plots display inhibition of IFNγ production by NK cells (y-axis) in presence of either isotype or αTRAIL as relative reduction compared to no antibody. Data information: Wilcoxon signed-rank test. B, Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.(C) Representative contour plots showing conjugate formation between NK cells and 721.221 target cells as determined by flow cytometry. Plots showing three distinct populations at the beginning of the co-culture (0 min) and after 50 min: single 721.221 cells (CT Orange-positive), single NK cells (CT Violet-positive) and conjugates (double-positive). Relative frequency of NK cells in conjugates is calculated based on the total number of gated NK cells. (D) Comparison of conjugate formation after co-culture between NK cells and 722.221 cells after 0 min and after 50 min in the presence or absence of 10 µg/ml αTRAIL or isotype control (n = 8 different donors). Left panel: Box plots showing relative frequency of NK cells in conjugates (y-axis) for different time points and conditions (x-axis). Right panel: Box plots display inhibition of conjugate formation (y-axis) in the presence of either isotype or αTRAIL as relative reduction compared to no antibody. Data information: Wilcoxon signed-rank test. D) Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.(E) Expression levels of phosphorylated signaling proteins. Upper panel: Concatenated contour plots of one donor depicting the expression of phosphorylated signaling proteins Syk, p38 MAPK, Akt and PLC-γ2 for the following culture conditions and controls (x-axis: left to right): FMO, PBS, isotype, αTRAIL, αNKp46, αNKG2D, αCD16. Lower panel: Bar graphs showing the percentage of p-Syk+, p-p38 MAPK+, p-Akt+ and p-PLC-γ2+ NK cells after 30 min of stimulation (n = 3 different donors). Bar graphs represent the median, whiskers display minimum and maximum data points.
Figure 7Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (A) Left panel: Representative contour plots showing depletion of 721.221 target cells in the presence of NK cells. Middle panel: Percentage of target cells remaining (y-axis) after co-culture with NK cells in the presence of either αTRAIL or isotype control, in reference to target cells kept alone. Right panel: Box plots displaying difference of target cells remaining (y-axis) between αTRAIL and isotype conditions displayed as p.p. (n = 12 different donors). Each data point represents the mean of at least 2 technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (B) Left panel: Representative contour plots showing the percentage of .221-DR4/5KO (control) and .221-Cas9 cells (target) in the presence or absence of NK cells. Middle panel: Ratio between .221-DR4/5KO and .221-Cas9 cells (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of .221-Cas9 cells displayed as % (n = 12 different donors). Each data point represents the mean of at least 3 technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (C) Left panel: Representative contour plots showing the percentage of Raji-pSIP (control) and Raji-DR5++ (target) in the presence or absence of NK cells. Middle panel: Ratio between Raji-pSIP and Raji-DR5++ (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of Raji-DR5++ cells displayed as % (n = 12 different donors). Each data point represents the mean of at least three technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.
Figure 2A. Immunoblot analysis of the levels of the indicated proteins in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.C. Immunoblot analysis of Klf4 and tubulin levels in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.H. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type and stat6-/- iBMDMs for 60 or 120 min.
Figure 3D. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and stat6-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments. E. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.
Figure 4A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min. B. Immunoblot analysis of Arg1and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min.F. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min. G. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min.
Figure 5A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min. B. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min.K. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf3-/- for 60 or 120 min. L Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf31-/- for 60 or 120 min.
Figure 6A. Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or tlr4-/- for 60 or 120 min. B. Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or myd88-/- for 60 or 120 min.D. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10-/- for 60 or 120 min.F. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10-/- for 60 or 120 min.L. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and il10-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments. M. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course in wild-type (WT) and il10-/- macrophages. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.
Figure 7A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the LPS O-polysaccharide mutant, strain 52145-Δglf, for 60 or 120 min.C. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-ΔwcaK2, for 60 or 120 min.
Figure 8A. arg1 mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h. D. ido mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h.B. il10 mRNA levels were assessed by qPCR in M-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h. C. chi3l mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h.E. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 for 60 or 120 minO. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-ΔwcaK2, for 60 or 120 min.
Figure 1.SARS-CoV-2.For weigh loss record, hACE2 mice (n=7) and WT mice (n=3) were experimentally challenged intranasally with SARS-CoV-2, and the ACE2-Mock mice (n=3) were used as control. And then the weight loss was recorded over 14 days (a, ANOVA, ***p<0.001).To screen virus replication, 12 mice were infected in each group, and 3 mice per group were sacrificed and their major organs harvested for viral load and virus titer at 1 dpi, 3 dpi, 5 dpi and 7 dpi respectively. The distribution of SARS-CoV-2 in the primary organs of ACE2-HB-01 mice was detected by qRT-PCR (b).Virus titers of lungs were determined on Vero E6 cells (c, unpaired t-test, ***p<0.001)the virus isolated from lungs of ACE2-HB-01 mice at 3 dpi was observed by electron microscope (d).The specific IgG against SARS-CoV-2 was detected at 21 dpi by ELISA (e, unpaired t-test, ***p<0.001).
Figure 2.mice.a. Gross pathology and histopathology of lungs from WT-HB-01 mice (3 dpi), ACE2-Mock mice (3 dpi) and ACE2-HB-01 mice (3 dpi and 5 dpi). Postmortem examinations showed focal dark red lesions (red arrow) throughout the dorsal of the right middle lobe of the lung at 3dpi. The lesions progressed into multifocally scattered-dark reddish purple areas and palpable nodules (red arrow) throughout the right lobe of the lung at 5 dpi. Histopathological observation indicated that moderate interstitial pneumonia with thickened alveolar septa (black arrows) and infiltration of lymphocytes (red arrows). The swollen and degenerative alveolar macrophages (green arrows) are scattered within the alveolar cavities at 3dpi and 5 dpi.b. Immunohistochemical examination of lungs of each group. The sequential sections were stained by HE and IHC, respectively. The viral antigens were observed in the alveolar macrophage (green arrows), the alveolar epithelia (blue arrows), and the degenerative and being desquamated bronchial epithelial cells (black arrows). Black bar = 100 µm, red bar = 50 µm.
Figure 3. Immunofluorescence analysis of viral antigens in lungs of SARS-CoV-2-infected hACE2 mice.Co-localization of SARS-CoV-2 S protein and hACE2 receptor in hACE2 mouse lungs, the sections were incubated with anti-SARS-CoV-2 S protein antibody, anti-human ACE2 antibody, and DAPI. The lung sections of ACE2-Mock mice (a-d). The lung sections of ACE2-HB-01 mice (e-h). The white arrows showed the viral S protein (f) and hACE2 (g), respectively, the yellow arrow showed the merge of viral S protein and hACE2 (h). White bar=25 µm.
Figure 1(C) Clonal cell lines expressing p53-Venus were engineered to monitor p53 expression dynamics ("input") and two canonically regulated p53 target promoters expressing mCherry ("output") in response to Nutlin-3 treatment.(D) Representative phase contrast and yellow fluorescent (indicating p53-Venus levels) images at the indicated time points for a cell exposed to the "natural dynamics" Nutlin-3 dosing regimen.(E-F) p53-Venus expression in response to the "natural dynamics" Nutlin-3 dosing regimen. Single cell traces (gray; E) and the mean (red; E) are shown for p53-Venus expression in response to each Nutlin-3 regimen. Heat map (F) shows an alternative representation of all traces as shown in (E). N = 51 cells.
Figure 2(A) Representative phase contrast, yellow fluorescent (indicating p53-Venus levels), and red fluorescent (indicating reporter mCherry levels) images at the indicated time points for MDM2 promoter reporter cells exposed to the "natural dynamics" Nutlin-3 dosing regimen.(B-G) Single cell traces of mCherry expression for the "responding" (light gray) or "not responding" (dark gray) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics (B), low amplitude (D), high amplitude (D), high frequency (E), low frequency/short duration (F), or long duration (G) Nutlin-3 dosing regimens. The average trace for responding and not responding cells is shown in red and blue, respectively. Heat maps show alternative representation of all single cell traces below each associated time course plot.
Figure 4(B-G) Effects of p53 duration modulation on target promoter activation in terms of the mean timing (B, E), magnitude (C, F), and rate (D, G) for the MDM2 (B-D) and CDKN1A (E-G) promoters in response to short (light green) and long (dark green) p53 durations. N = at least 45 cells per condition, error bars = SEM.(H) Mean nuclear MDM2-YFP levels in single cells at times corresponding to the first pulse (5.5 h) or second pulse (11 h) of p53 expression in response to natural and long duration Nutlin-3 dosing regimens. N = at least 40 cells per condition, error bars = SEM.
Figure 5(A) Effects of p53 frequency modulation on target promoter activation in terms of the mean timing (A, D), magnitude (B, E), and rate (C, F) for the MDM2 (A-C) and CDKN1A (D-F) promoters in response to low (light blue), natural (medium blue), high (dark blue) p53 frequencies. N = at least 45 cells per condition, error bars = SEM.(H) Percentage of responding cells for the MDM2 and CDKN1A promoters in response to low, natural, and high frequency p53 pulses.
Figure 6(A-B) Representative phase contrast, red fluorescence (indicating geminin-mCherry levels), and yellow fluorescence (indicating p53-Venus levels) images of cells in response to a low (A) or high (B) frequency Nutlin-3 dosing regimen over 40 h.(C) Geminin-mCherry traces in single cells treated with the low, natural, or high frequency Nutlin-3 dosing regimens. N = at least 50 cells per condition.Percentage of cells undergoing cell division (D) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimensPercentage of cells positive for geminin-mCherry expression (E) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens
Figure 1A Representative immunofluorescence for PH3 (green), combined with DAPI staining (white), of E14.5 wildtype (WT) and 11B mouse dorsolateral neocortex rostrally. B Representative immunofluorescence for pVim (yellow), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (50 µm x 65 µm) in (B) indicate areas that are shown at higher magnification in (B'). Arrow, pVim+ BP with basal process (mitotic bRG); notched arrowheads, basal process; arrowhead, pVim+ BP without basal process (mitotic bIP). C-E Quantification of PH3+ APs (C), PH3+ BPs (D) and pVim+ bRG (E left) and bIPs (E right) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Note the difference in ordinate scales in (E). F Data information: Images are single optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0269 (D); P = 0.0302 (E, left); P = 0.0139 (E, right)F Representative immunofluorescence for Pax6 (cyan) and Tbr2 (magenta), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (25 µm x 25 µm) in (F) indicate areas that are shown at higher magnification in (F'). Outlined by dashed white lines, Pax6+ & Tbr2+ BP; outlined by solid white lines, Pax6+ & Tbr2- BP. G-K Quantification of Pax6+ & Tbr2- cells in VZ (G), Pax6+ & Tbr2+ cells in VZ (H), Pax6+ & Tbr2- cells in SVZ and IZ (I), Pax6+ & Tbr2+ cells in SVZ and IZ (J) and Pax6- & Tbr2+ cells in SVZ and IZ (K) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. L Data information: Images are Z projections of 4 optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0281 (I); P = 0.0088 (J); P = 0.0014 (K)L-N, Sequential BrdU-EdU labelling. BrdU was injected into pregnant mice at E13.5, i.e. 24 hr before sacrifice at E14.5, and EdU was injected into the same pregnant mice 0.5 hr before sacrifice at E14.5. (L) Representative immunofluorescence for BrdU (magenta), combined with EdU staining (cyan) and DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. (M, N) Quantification of BrdU+ & EdU+ cells (M) and the percentage of BrdU+ cells that are BrdU+ & EdU+ (N) in VZ and SVZ in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Data information: Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0049 (M, SVZ); P = 0.0323 (N, SVZ).
Figure 2H Representative immunofluorescence for Tbr1 (cyan), Ctip2 (magenta) and Satb2 (green), combined with DAPI staining (white), of E18.5 WT and 11B mouse neocortex at the position where cortical thickness was measured (A, red lines). I Quantification of zone thickness of E18.5 WT (white) and 11B (black) mouse neocortex at the position where cortical thickness was measured (A, red lines). J Data information: Images are single optical sections Scale bar, 20 µm Data are the mean of 7 (WT) and 8-12 (11B) littermate embryos, which were derived from 4 separate litters. Error bars indicate SD. Two-way ANOVA, followed by Bonferroni's multiple comparisons test (B-D); two-tailed unpaired Student's t-test P = 0.0287 (I, IZ); P = 0.0039
Figure 3A Dorsal view of adult wildtype (WT) and 11B mouse brain at P56. Orange lines illustrate the measurements of neocortex width and length; red lines illustrate the measurements of neocortex area. B, C Quantification of neocortex width, length and area. D Data information: Scale bar, 1 mm Data are the mean of 8 (WT) and 8 (11B) adult littermate mice. Error bars indicate SD, two-tailed unpaired Student's t-test , * P < 0.05, ** P < 0.01, **** P < 0.0001. P < 0.0001 (B, neocortex width); P < 0.0001 (C)G, H Representative immunofluorescence for Ctip2 (magenta), Satb2 (green) and Brn2 (yellow), combined with DAPI staining (white), of P56 adult WT and 11B mouse neocortex at the position where cortical thickness was measured (D, red lines). Data information: Images are single optical sections Scale bar, 20 µm
Figure 4IntelliCage was used to evaluate the memory flexibility of adult 11B mice. Four separate sessions were carried out: (i) 5 WT males together with 5 11B males; (ii) 5 other WT males together with 5 other 11B males; (iii) 5 WT females together with 7 11B females; (iv) 4 other WT females together with 8 other 11B females. Data are the mean of the total 10 males (WT), 10 males (11B), 9 females (WT) and 15 females (11B) adult littermate mice. Data points were obtained at consecutive days, error bars indicate SD. (A, B) Quantification of the discrimination error rate in the first 100 visits during the drinking phase of the acquisition stage of adult wildtype (WT) and 11B mice (male, A; female, B) in the IntelliCage. Overall place learning performance of WT and 11B mice was analysed using two-way ANOVA followed by Fisher's LSD test; all data points during the acquisition phase were included. For males (A), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. For females (B), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. Note that (i) the two male sessions and the two female sessions yielded essentially the same results, and (ii) not shown in panels the two separate male sessions yielded essentially the same results, and the two separate female sessions yielded essentially the same results.
Figure 1Quantification of NANOG-positive iPSC colonies obtained after 14 days of OSKM induction in MEFs treated with either non-target or Kdm3b siRNA in FBS media. Values represent fold change over non-target control providing relative quantitation. Quantification of NANOG-positive iPSC colonies obtained after 12 or 14 days of OSKM induction in MEFs treated with either non-targeting or Kdm3b siRNA in KSR media. Values represent fold change over non-target control providing relative quantitation.Quantification of NANOG positive colonies after retroviral expression of OSKM (Left) or OSK (Right) in MEF treated with non-targeting or siRNA targeting Kdm3. Nanog counts were performed on Day 16 for OSKM or Day 21 for OSK reprogramming. Values represent fold change over non-target control providing relative quantitation.Immunoblot of H3K9me1/2 histone demethylase in WT or Kdm3b CRISPR targeted pre-iPSC clones. These pre-iPSCs carry a Nanog-GFP reporter. Parent line (WT), and two Kdm3b-KO clones using gRNA targeting either exon 1 (KO#1) or exon 2 (KO#2) are shown. RNA Polymerase II levels are the loading control.Quantification of Nanog-GFP positive cells after 10 days of AA+2i treatment of WT or Kdm3b-deleted pre-iPSCs.Immunoblot of H3K9 methylation levels upon addition of H3K9 methyltransferase inhibitor UNC0638 in WT or Kdm3b-KO pre-iPSCs.Quantification of percent Nanog-GFP positive cells after 10 days of AA+2i or AA+2i with 5µM UNC0638 in WT or Kdm3b deleted pre-iPSCs.