EMBO/soda-nel huggingface dataset:
KAP1 regulates ERVs in adult human cells and contributes to innate immune control (2018)
Figure 1

Figure 1. The human retrovirus HERVK14C is repressed by KAP1 in undifferentiated and differentiated cells including adult PBMCs

(A) Schematic diagram showing the age of HERVK14C. (B) Venn diagram showing the chromosome copies detected by the two primer sets. (C) qRT-PCR expression of endogenous repeats following shRNA-mediated KAP1 depletion in NTERA-2 cells. Results were normalized to β2 microglobulin (B2M). KAP1 expression levels were verified by qRT-PCR and Western blot. A representative experiment of 2 experiments is shown. Two-tailed unpaired t tests were done (HERVK14C_2 p-value = 0.002300). (D and E) qRT-PCR expression of endogenous repeats following KAP1 knockout in HeLa (D) and 293T cells (E). Results were normalized to B2M. KAP1 expression levels were verified by qRT-PCR and Western blot. For (E), we found that clone I (also treated with KAP1 sgRNA) retained KAP1 protein expression (E, right panel) so we only used clones II, III and IV to explore phenotype (E, left panel), which represented validated KAP1 knockouts. Two-tailed unpaired t tests were done and p-values are: (D) HERVK14C_1: 0.0017, HERVK14C_2: 0.0044, SVA D VNTR: 0.0243 (E) HERVK14C_2: <0.0001. Clones 8, 12 and 15 (from D) were selected for mRNA-sequencing. (F) qRT-PCR expression of endogenous repeats following shRNA-mediated KAP1 depletion in PBMCs (day 6 post-transduction). Results were normalized to B2M. All error bars show standard deviation (SD). All numbers above bars depict fold changes compared to control cells (to one decimal place).
KAP1 regulates ERVs in adult human cells and contributes to innate immune control (2018)
Figure 2

Figure 2. A common role for KAP1 in repressing ERVs and ZNFs through H3K9me3

(A) Boxplots showing repeats significantly upregulated (>2 fold where p=<0.05 using DESeq2) in knockout (N=3) compared to wildtype (N=3) HeLa cells based on mRNA-sequencing data. P values = 0.0174 (HERV-S), 0.0047 (HERV-K14C), 0.00013 (HERV-T LTR6B) and 1.90E-06 (HERV-T HERVS71). HERV-T and HERV-S but not HERVK14C also reached significance when only adjusted p-values were considered, where differences are compared to the whole of repBase. KAP1 binding (ENCODE data) is shown in Fig S1A. All numbers above bars depict fold changes compared to control cells (to one decimal place).

(B) Evolutionary tree showing the age of KAP1-repressed HERVs identified in A). Here, HERV-K refers to HERVK14C. Estimated ages of stated lineages are given and marked with a star. Myr: million years (C) The 38 upregulated genes identified (>2 fold where padj=<0.05) were converted to DAVID IDs and used for gene ontology analysis. 3 gene clusters were significantly enriched (p-value = <0.05, drawn on the plot as a dotted line) in the data set: zinc finger proteins (p=2.2 x 10-7), negative regulators of metabolic processes (p=0.0036) and angiogenesis (p=0.011). Venn diagrams on the right show numbers of upregulated genes, ZNFs and KZNFs and the overlap. (D) The nature of conserved KAP1 binding sites between human ESCs [11] and 293Ts (ENCODE) (see Fig EV2D) is shown (left pie chart) compared to their abundance in the genome (right pie chart). (E) The 614 KAP1 common binding sites (see Fig EV2D) were interrogated for their nearest gene and this gene list was converted to DAVID IDs and used for gene ontology analysis. 4 gene clusters were significantly enriched (p-value <0.05, drawn on the plot as a dotted line): zinc finger proteins (p=1.1 x 10-19), fibronectin folding (p=0.016), protein phosphatase (p=0.033), and synapse (p=0.047). Venn diagrams on the right show numbers of KAP1-bound loci, ZNFs and KZNFs and the overlap. (F) Genomic coordinates of the common KAP1 sites identified in Fig EV3D were subjected to ChIP-seq correlation analyses using ChIP-Cor software (see methods). Each plot shows duplicate ChIP-seq experiments from ENCODE. See Fig EV3B for complete data.
KAP1 regulates ERVs in adult human cells and contributes to innate immune control (2018)
Figure 3

Figure 3. PBS-dependent de novo repression of ERVs is conserved in primary cells

(A) Schematic diagram showing reporter constructs used. The 18-bp PBS sequence was cloned upstream of the HERVK14C LTR promoter (which is identical in all vectors) in an antisense orientation. (B) NTERA-2 cells were transduced with either an empty vector (WT) or the same vector containing an shRNA against KAP1 (KD) prior to puromycin selection and transduction with increasing doses of GFP reporter vectors. GFP expression was analysed 72 hours post reporter transduction. A representative experiment of 2 experiments is shown. Two-tailed unpaired t test p-values: PBS-LTR-GFP: 0.0148; PBSChr15-LTR-GFP: 0.0377 (C) The same as (B) except that here KAP1 wildtype (WT) and knockout (KO) 293T cells were used. P-values: PBS-LTR-GFP: 0.0044; PBSChr15-LTR-GFP: 0.0077. (D) PBMCs were transduced with increasing doses of GFP reporter vectors (vectors were normalized to the same number of infectious units after titering them on permissive cells), left plot. Vectors were integrated at similar levels as measured by GFP Taqman qPCR, right plot. A 293T cell line with a single vector copy integrant (PGK-GFP) was used as a control to estimate the absolute copy numbers. A representative experiment of 3 experiments is shown here. Two-tailed unpaired t test p-value 0.0288 (dose 3). Error bars show SD. (E) The expression of 340 KZNFs was assessed in multiple cell types using public mRNA-sequencing data and Venn diagrams show the overlap in expression profiles. KZNFs were considered to be expressed in a particular cell type if their expression value was >0.5 RPKM in all replicates.
KAP1 regulates ERVs in adult human cells and contributes to innate immune control (2018)
Figure 4

Figure 4. Cytosine methylation acts on KAP1-regulated retrotransposons and prevents innate immune activation

(A) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following shRNA-mediated KAP1 depletion in HeLa cells (day 6 post transduction), normalized to B2M and GAPDH respectively. n=2 and two-tailed unpaired t test p-values are: HERVK14C: 0.025, 0.003; SVA VNTR: 0.00027, 0.01; ISG56: 0.002; CCL5: 0.007, 0.00003; CXCL10: 0.0008; Mxa: 0.011; IKba: 0.008, 0.01. See also Fig S2AB. (B) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following 5-AZA treatment of HeLa cells and PBMCs (day 6 post transduction). A representative experiment of two is shown in each case. Two-tailed unpaired t tests were performed. HeLa cell p-values: ISG56: 0.005; CCL5: 0.0006; CXCL10: 0.0007; Mxa: 0.0001; IFNb: 0.02. For repeat expression (left panel), p values not available as N=2. PBMC p-values: HERVK14C: 0.1; SVA D VNTR: 0.003; ISG56: 0.001; CCL5: 0.012; CXCL10: 0.012; Mxa: 0.02; IKba: 0.08. Results were normalized to GAPDH and B2M (GAPDH results shown). See also Fig S2C for results in 293T cells. All error bars show SD. (C) DNA methylation at endogenous SVAs was measured over 18 CpGs in the stated treatment groups (day 5 post transductions, or day 2 post 5-AZA treatment). Each point represents the average methylation state of one sequence with at least 10 sequences analyzed per group. Mann-Whitney tests were performed to compare shControl to shKAP1 (p=0.6593) and DMSO to 5-AZA (0.0027). 5-AZA was added to all experiments at 7uM. All numbers above bars depict fold changes compared to control cells (to one decimal place).
KAP1 regulates ERVs in adult human cells and contributes to innate immune control (2018)
Figure 5

Figure 5. KAP1-depletion leads to a decrease in H3K9me3 at ERVs and a MAVS-dependent innate immune response

(A) H3K9me3 ChIP-PCR where error bars show SD of 3 independent IPs. Results show the IP enrichment relative to the total input normalized to GAPDH as a negative control region. IgG control IPs gave only background enrichments, shown. (B) 293T cells were transduced with the stated shRNAs and cells harvested 4 days after puro selection for Western Blot to verify KAP1 and SETDB1-depletion (left) and ChIP (right). Cell numbers were normalized per treatment group before sonication and IPs were performed in triplicate. Results show IP relative to total input and GAPDH normalized. IgG control IPs gave only background enrichments shown. One representative experiment of two is shown. Unpaired t tests were performed. P values: ZNF239: KAP1, 0.0292; SETDB1, 0.0137; HERVK14C_1: KAP1, 0.0227; SETDB1, 0.0082; HERVK14C_2: SETDB1, 0.0462; SVA D VNTR, SETDB1, 0.0064. The inlay shows qRTPCR expression analysis of HERVK14C in the same experiment, normalized to B2M or GAPDH as stated. All error bars show SD. (C) Venn diagram showing H3K9me3 enriched sites (18271) as those identified in both ChIP replicates in the human cell line K562 (left). Right: % of ISGs or random genes intersecting a H3K9me3 or KAP1 ChIP-seq peak. 5kb upstream and downstream of each gene was also included. N=437 genes per group. (D) An IFIT1 promoter-based Luciferase reporter THP-1 cell line was used or 293T cells or HeLa cells transduced with an IFN-β promoter-based Luciferase reporter. These cell lines were transduced with an shKAP1 or shControl vector or a vFLIP-expressing vector and luciferase measured 6 days later. Error bars show SD. Unpaired t tests were used to compare shControl to shKAP1 samples: p values = 0.122 (THP-1), 0.0001 (HeLa), 0.0001 (293T cells). (E) WT, STING KO and MAVS KO THP-1 cells were verified by Licor blot. (F) Cells from (E) were untreated or transduced with shControl or shKAP1 vectors and Luciferase read over time (left). Cells were also treated with control stimulants (right). Results show the average Luciferase secreted over two time points (days 5 and 6 for the left plot and days 1 and 2 for the right plot) with all error bars showing SEM. HT-DNA: herring testis DNA.
PINK1 Phosphorylates Drp1 S616 to Regulate Mitophagy-independent
Mitochondrial Dynamics (2020)
Figure 2

Figure 2. Drp1, but not Drp1S616A, reverse abnormal dendritic mitochondrial fission in PINK1-null mouse neurons.

(a, b). Decreased Drp1S616 phosphorylation in PINK1-null primary neurons. Lysates of DIV6 neurons generated from PINK1KO and PINK1WT control mice were immune-detected with antibodies against either phospho (Ser616)-Drp1 (pS616) or Drp1. Tuj1 was detected as a loading control (a). Quantitation of pS616/Drp1 for each experiment is shown (b). Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.

(c, d). TEM analysis of mitochondria in mouse substantial nigra. Sections of substantial nigra from 18-month-old PINK1KO and PINK1WT control mice were analyzed under TEM (c, left panels, scale bar=2 µm). Amplified images of mitochondria from dot line frames gated region are shown (c, right panels, scale bar=1 µm). Asterisks indicate abnormal elongated mitochondria. A quantitative analysis of mitochondria with perimeter >2µm is shown (d). Over 300 identifiable mitochondria (cristae and/or double membrane) per mice were randomly and blindly selected for perimeter measurement. Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.

(e, f). Overexpression of Drp1WT, but not phosphorylation mutant Drp1S616A, suppresses mitochondrial fusion in dendrites of PINK1KO primary neurons.

(e). DIV4 neurons generated from PINK1WT control or PINK1KO mice were co-transfected mito-GFP with either pcDNA3.1 (3.1) or Drp1WT or Drp1S616A. Neurons were immuno-detected with an antibody against myc-tag (Myc) (red, to detect exogenous Drp1). Mito-GFP: green, to detect mitochondria. Representative images were shown, scale bar=25 μm. Magnified dendritic shafts gated by a dot line frame are shown (Zoom, right panels, scale bar=5 μm).(f). Quantification of Mito-index in dendrites of neurons from experiments of (e). 10 neurons were randomly selected and analyzed in each experiment. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.
PINK1 Phosphorylates Drp1 S616 to Regulate Mitophagy-independent
Mitochondrial Dynamics (2020)
Figure 4

Figure 4. PINK1 phosphorylates Drp1S616 to regulate mitochondrial fission that is independent of mitophagy.

(a-e). ATG5 is dispensable for PINK1-mediated mitochondrial fission. MEF cells derived from ATG5-null (ATG5KO) and their wildtype control mice (ATG5WT) were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either PINK1WT or a kinase-dead mutant PINK1D384N. Cells were induced with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images are shown, scale bar=25 μm (a). Higher magnification images are also included (panels beneath the large cell images, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified (b-e). Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with either 20 μM CCCP for 2 hours (f) or 150 μM actinonin for 6 hours (h), followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.
PINK1 Phosphorylates Drp1 S616 to Regulate Mitophagy-independent
Mitochondrial Dynamics (2020)
Figure 6

Figure 6. Drp1 rescues PINK1KO induced crushed thorax and mitochondrial abnormality in dATG7-deficient Drosophila.

(a, b). Drp1 rescues PINK1KO induced collapsed thorax phenotype in

dATG7-deficient Drosophila. Representative SEM images of thorax from PINK1WT, (upper panels) and PINK1KO (lower panels) flies combined with dATP7 deficiency (dATG7KO) or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1 (Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D (Mhc>hDrp1S616D) are shown (a). Quantitation of crushed thorax in flies with indicated genotypes is presented (b). > 100 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.

(c, d) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila. Representative TEM images of indirect flight muscle from PINK1WT (upper panels) and PINK1KO (lower panels) flies combined with dATG7KO or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1(Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A(Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D(Mhc>hDrp1S616D) are shown (c). Scale bar=1 μm. Normal and abnormal mitochondrial number per mm2 from each experimental group were and quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (d).

(e). Drp1 rescues PINK1KO induced ATP reduction in Drosophila. ATP contents of thorax muscle tissues from the indicated genotypes were measured and normalized against the protein levels One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.
PINK1 Phosphorylates Drp1 S616 to Regulate Mitophagy-independent
Mitochondrial Dynamics (2020)
Figure 7

Figure 7. Decreased Drp1S616 phosphorylation in human fibroblasts derived from PD patients harboring PINK1 mutations.

(a-f). Pedigrees of PD families with PINK1 mutations and Sanger sequencing conformation. Pedigrees of three families with four PD patients (a, c, e) and corresponding sequencing chromatogram of four PD patients harboring PINK1 mutations (b, d, f) are shown. Red arrows indicate the mutations in each patient.

(g, h). Reduction of Drp1S616 phosphorylation in dermal fibroblasts derived from PD patients with PINK1 mutations. Lysates of cells derived from three unrelated normal control individuals (Control, C) and four familial PD patients with PINK1 mutations (PINK1mutant) were immunoblotted with antibodies recognizing either phosphor-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (g). A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) is shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.

(i, j). Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients. Lysates of cells derived from five unrelated normal control individuals (Control, C) and seven unrelated sporadic PD patients (Sporadic PD, SP) were immunoblotted with antibodies recognizing either phospho-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (i). A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) in sporadic cases is shown (j). Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.
Receptor interacting protein kinase 2–mediated mitophagy regulates inflammasome activation during virus infection (2013)
figf1

(a) Survival rate of wild-type mice (WT) and indicated knockout mice after intranasal infection with 750 plaque forming units (PFU) of the PR8 virus. (b) Hematoxylin and eosin (H&E)-stained lung sections from mice infected with PR8 virus on day 2. Arrows indicate cellular infiltrates and necrotic debris occluding the airways. Original magnification, ×40. (c) Disease severity ('lung score') and extent of cell death (necrosis score) on day 2 after infection based on analysis of H&E-stained lung sections. (d) Necrosis score on day 7, estimated visually from H&E-stained lung sections, and flow cytometry analysis for annexin V–stained lung cell populations. (e) Immunohistochemistry staining of neutrophils occluding the airways of PR8-infected mice. Original magnification, ×20. (f,g) Flow cytometry analysis of neutrophil, dendritic cell (DC) and macrophage cell numbers on days 2 and 7. (h) Pulmonary viral titers on days 2 and 7 after infection in Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (log-rank test (a) and unpaired two-sided Student's t-test (c,d,f,g). Data are pooled from three experiments (a,f–h; mean ± s.e.m. in f–h; n = 10–12 mice total) or are representative of three experiments (b–e; mean ± s.e.m.; n = 3–5 mice per experiment).
Receptor interacting protein kinase 2–mediated mitophagy regulates inflammasome activation during virus infection (2013)
figf2

(a–c) Supernatants of whole lung homogenates taken on days 2 and 7 after infection of wild-type mice (WT) and Ripk2−/− mice with the PR8 virus were analyzed for IL-6 and TNF (day 2; a), IL-18 and IFN-γ (b), and indicated chemokines (c). *P 0.05, **P 0.01 (unpaired two-sided Student's t-test). Data were normalized for total protein (t.p.) from lung homogenates. Data are pooled from three experiments (mean ± s.e.m.; n = 10–12 mice total).
Receptor interacting protein kinase 2–mediated mitophagy regulates inflammasome activation during virus infection (2013)
figf3

(a–c) Analysis on day 2 after infection with PR8 virus of wild-type mice (WT) or Ripk2−/− mice injected intraperitoneally with control rabbit serum and Ripk2−/− mice injected intraperitoneally with anti–IL-18 neutralizing antiserum (αIL-18Ab) 3 h before infection, for IL-18, IFN-γ and CCL2 amounts in whole lung homogenates (a; t.p., total protein), and for neutrophil numbers by immunohistochemistry (b) and by flow cytometry (c). Original magnification, ×20. (d) Analysis of survival of indicated mice. (e) Neutrophil numbers in WT, Ripk2−/− and Ripk2−/−Il18−/− mice on day 2 after PR8 infection. (f) Survival of Ripk2−/−Il18−/−, Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (one-way ANOVA with Tukey's post-hoc test (a,c,e) and log-rank test (d,f)). Data are representative of two independent experiments (mean ± s.e.m.; n = 6–8 mice per experiment).
Receptor interacting protein kinase 2–mediated mitophagy regulates inflammasome activation during virus infection (2013)
figf4

(a,b) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL-18 production by using IL-18 immunohistochemistry (a) and IL-18 ELISA (b) of whole lung homogenates. WT, wild type; t.p., total protein. In a, blue arrows denote IL-18+ macrophages, and red arrows denote IL-18+ bronchial epithelium. Original magnification, ×200. (c) Flow cytometry analysis of NK cell counts in Ripk2−/− and wild-type mice. (d) Analysis of wild-type and Ripk2−/− NK cells for IFN-γ production on a per cell basis. (e) Accumulation of T and B cells in lungs. (f) Analysis of CD8+ T cells for IFN-γ and CD44 expression in wild-type and Ripk2−/− mice. *P 0.05, **P 0.01 (one-way ANOVA with Tukey's post-hoc test (b) and unpaired two-sided Student's t-test (d,f)). Data are representative of two experiments (a,b,f) or three experiments (c–e) (mean ± s.e.m.; n = 6–8 mice per experiment).
Receptor interacting protein kinase 2–mediated mitophagy regulates inflammasome activation during virus infection (2013)
figf5

(a) Immunoblots showing active caspase-1 (casp-1) p20 compared to GAPDH loading controls in wild-type (WT), Ripk2−/− and Nod2−/− BMDCs after PR8 infection. (b) IL-18 release in PR8-infected WT, Ripk2−/− and Nod2−/− BMDCs. (c,d) Immunoblot analysis showing active casp-1 p20 (c) and IL-18 production (d) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10. (e,f) IL-18 and casp-1 activation in Ripk2−/− BMDCs treated with the NLRP3 inflammasome–specific inhibitor glyburide (glyb.) during PR8 infection. (g) Immunoblot analysis of casp-1 p20 during Listeria infection of WT and Aim2−/− BMDCs. *P 0.01, **P 0.001 (one-way ANOVA with Tukey's post-hoc test). Data are representative of six experiments (a,b) or three experiments (c–g) (mean ± s.d.; n = 2–3 replicates per experiment).
Receptor interacting protein kinase 2–mediated mitophagy regulates inflammasome activation during virus infection (2013)
figf6

(a) Immunoblot analysis of wild-type (WT), Ripk2−/− and Nod2−/− BMDCs for LC3-II (marker for autophagy) after 18 h of PR8 infection. (b,c) Numbers of LC3-II+ puncta (b), counted in at least 100 cells from five random fields from confocal images (c) of BMDCs after PR8 infection and/or chloroquine treatment. Original magnification, ×400. (d,e) Immunoblots of LC3-II and caspase-1 (casp-1; e) and IL-18 production (d) in wild-type and Ripk2−/− BMDCs after treatment with rapamycin (Rap). (f) Immunoblot analysis for LC3-II in lung lysates of PR8-infected wild-type and Ripk2−/− mice (1–5 indicate individual mice). (g,h) Lung neutrophil infiltration (g), and IL-18 and IFN-γ amounts (h) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)). Data are representative of six experiments (a,d,e; mean ± s.d.; n = 2–3 replicates per experiment), three experiments (b,c; mean ± s.d.; n = 2–3 replicates per experiment) or two experiments (f–h; mean ± s.e.m.; n = 5–7 mice per experiment).