EMBO/SourceData huggingface dataset:
Figure 3Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type 4 Tmem106b-/- mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed mRNA of Tmem106b-/- mice in comparison to wild type mice. From 622 detected genes 21 are significantly upregulated, while 12 are significantly reduced.Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type, 4 Grn-/ mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (B) Comparison of differentially expressed mRNA in Grn-/- and wild type mice. From 622 detected genes 32 are significantly upregulated, while only 1 is significantly reduced.Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type 5 Grn-/-/Tmem106b-/- mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (C) Volcano plot of differentially expressed brain mRNAs from Grn-/-/Tmem106b-/- mice in comparison to wild type mice. From 622 detected genes 110 are significantly upregulated, while 16 are significantly reduced.D. Display of differently expressed genes overlapping between the analyzed genotypes from (A-C). Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20% change in at least one mouse model. Note that 19 genes are significantly altered in all 3 genotypes in comparison to the wild type. 17 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 17 significantly altered genes. Data represent the mean ± SD.
Figure 4Western blot analysis of GFAP and IBA1 in total brain lysates from 4.5-month-old mice with the given genotype. Quantification of A. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.Expression of GFAP in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; MY = Medulla). Expression of IBA1 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; MY = Medulla). Scale bar indicates 100 µm.In vivo 18F-GE180 TSPO μPET imaging. Two female Grn-/-/Tmem106b-/- and five female wild type mice underwent longitudinal TSPO μPET at 2.0 and 3.5 months of age. All analyses were performed by PMOD (V3.5, PMOD technologies). Normalization of injected activity was performed by the previously validated myocardium correction method.
Figure 5Western blot analysis of CD68, CLEC7A, TREM2 and ApoE in total brain lysates from 4.5-month-old mice with the given genotype. Asterisk indicate unspecific band. (n = 3 biological replicates per genotype). Quantification of A. Protein expression was normalized to levels in wild type animals.Expression of CD68 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (TH = Thalamus). Expression of CLEC7A and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Expression of TREM2 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Scale bar indicates 25 µm.
Figure 6Western blot analysis of cathepsin (Cat) D, B, and L in total brain lysates from 4.5-month-old mice with the indicated genotype. Different maturation variants are indicated (p = preform, sc = single chain, hc = heavy chain) (n = 3 biological replicates per genotype). Quantification of proteolytically generated variants of cathepsin D, B, and L normalized to levels in wild type animals from (A).Catalytic activity of cathepsin D, B, and L. Total brain lysates from 4.5-month-old mice were incubated with quenched fluorogenic substrate for Cat D, Cat B and Cat L. The linear increase of fluorescence signal was measured and then normalized to wildtype. Data represent the mean normalized activity ± SD. (n = 3 biological replicates per genotype).
Figure 7Immunofluorescence analysis of lipofuscin (red) and IBA1 (green) in sagittal brain sections. Representative images of the Thalamus (TH). Scale bar indicates 25 µm.Western blot analysis of ubiquitin and the autophagy marker LC3 in RIPA, and p62 in RIPA and urea total brain lysates from 4.5-month-old mice with the indicated genotype. Calnexin used as a loading control. (n = 3 biological replicates per genotype). Quantification of B. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.Immunofluorescence analysis of p62 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; Medulla = MY). Scale bar indicates 100 µm.Localization of p62 mainly in NeuN positive cells in the cortex (CTX) of 4.5-month-old mice. Minor overlap of aggregates with microglial marker IBA1. Scale bar indicates 15 µm.Complete overlap of co-staining of p62 with NeuN in the cortex (CTX) and not with the reactive astrocycte marker GFAP. Scale bar indicates 15 µm.
Figure 8Immunofluorescence analysis of TDP-43 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; MB = Midbrain). White arrow heads indicate TDP-43 aggregation. Scale bar indicates 100 µm. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain, scale bar zoom in indicates 50 μm. (Animals 4.5 months of age).Immunofluorescence analysis of total TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sagittal brain sections by confocal microscopy. Representative images of one confocal layer in the midbrain. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain. Scale bar indicates 25 µm. (Animals 4.5 months of age).Western blot analysis of TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sequential high salt (HS)/RIPA/Urea fractionations of total brain lysates from 4.5-month-old mice with the indicated genotype. Analysis of C-terminal fragments of TDP-43 (CTF) in RIPA fraction. p-TDP-43 analyzed in Urea fraction. Quantification of (C) Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.
Figure 1, B. survival of Ssrp1fl/fl; CreERT2+/+ mice of different ages (weeks) after the start of tamoxifen (TMX) treatment. B. Kaplan-Meier survival curves. Log-rank test p = 0.032 for the oldest male age group versus all other male groups; p = 0.013 for all females versus all males; for all other comparisons p > 0.05. n = 6-10 mice.C. Normalized blood cell counts in Ssrp1fl/fl; CreERT2+/+ mice treated with vehicle or tamoxifen on day 7 after start of treatment. Mean ± SD, * indicates p < 0.05 (unpaired t-test). n = 3 mice.
Figure 2A. H&E staining of mouse organ sections. Scale bars: spleen, colon 100 μM, bone marrow - 50 μM.
Figure 4D. Fold-change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen-treated versus vehicle-treated mice. Cell types are identified by color as in panel B.
Figure 5D. UMAP plots with color-coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes.
Figure 6A, B. Effect of 4-OHT administration on colony formation by bone marrow cells of different genotypes. A. Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4-OHT. B. Quantitation of bone marrow colony assay. Mean colony number per plate ± SD, n = 3 plates per mouse. P values < 0.05 are shown (unpaired t-test). 2 mice of each genotype were analyzed. C, D. Growth of intestinal organoids in the presence or absence of 4-OHT. C. Photographs of small intestine organoids on different days after plating. D. Number of organoids in the presence and absence of 4-OHT. Mean fold change between number of organoids in control and 4-OHT treated wells ± SD, n = 4 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from one mouse of each genotype in two independent experiments.F, G. Effect of 4-OHT administration on intestinal organoids of different genotypes kept in the presence (undifferentiated) or absence (differentiated) of R-spondin-1. F. The viability of organoids assessed by the resorufin assay. Mean ± SD, n = 3 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from two mice of each genotype. G. IF images of intestinal organoids from Ssrp1fl/fl; CreERT2+/+ mice kept in the presence or absence of R-Spondin-1. The organoids were stained with antibodies to LGR5 and SSRP1 and counterstained with Hoechst (DNA).Effect of 4-OHT administration on MSCs and their differentiated counterparts. H. Methylene blue viability assay for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days and then treated with vehicle or 4-OHT. Mean ± SD, n = 3 plates per mouse. P < 0.05 are shown (unpaired t-test). MSCs were isolated from two mice of each genotype.
Figure 7A. EU incorporation measured by flow cytometry. Histograms of EU fluorescent signals from two biological replicates for each condition (except no EU). B. SSRP1 staining of the cells in A.
Figure 8G. Box plots of mean read density (y-axis) at the gene body (bins 21-100 out of 100 between TSS - TES) for all genes and genes expressed at different levels. *** - p < 1e-10 (paired t-test). n = 2 biological replicates. Central band is mean, box is interquartile range, whiskers are minimum and maximum.H. Comparison of Hoechst 33342 binding to live control and 4-OHT-treated MSCs from different genotypes. Data are presented as the median fluorescence intensity ± SD, n = 4 biological replicates. P < 0.05 is shown (unpaired t-test).
Figure 2(F) Representative images of GFP-positive HEK293 cells at day 7 post-transduction using 1x106 TU/cell of RGD4C.TPA.GFP or RGD4C.AAVP.GFP particles. Non-targeted (NT) particles were included as control. Data shown are representative of five independent experiments, performed each in triplicates. Scale bar, 100 μm.
Figure 3(A) Quantification of gene expression in HEK293 cells after transduction with 0.5x106, 1x106, and 2x106 TU/cell of RGD4C.TPA, RGD4C.AAVP or non-targeted (NT) particles carrying Lucia. Luciferase activity was measured at days 1 to 6 post-transduction. Data shown are representative of three independent experiments (n = 3), and are expressed as mean + SEM. One-way ANOVA with Tukey's HSD test was used for data analysis.(B) Representative images of FITC-labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post-inoculation (n = 3). Scale bar, 100 μm. (C) Particle diffusion using a vertical trans-well system partitioned by Matrigel. Samples were collected from the wells below the Matrigel at days 1 to 7, n = 3, technical replicates. Data are expressed as mean + SEM, independent t-test was used for data analysis.(D) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins, then quantified by FACS at 2 and 4 hours post-transduction, 0.1X106TU/cell, and normalised to particle length. Data are expressed as mean + SEM of n = 3 independent experiments, independent t-test was used for data analysis.(E) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post-transduction with 0.5x106 TU/cell of RGD4C.TPA and RGD4C.AAVP, using qPCR with primers specific to AAV-2 ITRs. Data are representative of n = 3 independent experiments and expressed as mean + SEM, independent t-test was used for data analysis.(F) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma-derived neural stem cells with 1x106 TU/cell. Data are expressed as mean + SEM of n = 3 independent experiments. One-way ANOVA with Tukey's HSD test was used for data analysis.(G) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia. Experiments were repeated three times and performed in triplicates. Data are shown as mean + SEM and are representative of one experiment. Independent t-test was used for data analysis.
Figure 4(B) ELISA quantification of TNFα production in media collected on days 3 to 5 post-transduction of human primary GBM001 tumor spheres at 1x106 TU/cell of targeted (RGD4C) or non-targeted (NT) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis.(C) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C.TPA.TNFαIL2 to NOD/SCID mice bearing subcutaneous human GBM001. Tumors and healthy organs were harvested at day 18 post-transduction and analysed using RT-qPCR to quantify the TNFαIL2 transcripts. Data shown are expressed as mean + SEM, from n = 3 mice. Two-way ANOVA was used for data analysis.(D) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene, at day 18 post targeted TNFαIL2 cytokine treatment. (E) Analysis of tumor growth in representative mice, from D, using the tumor bioluminescence data (total flux) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0.
Figure 5(B) Investigation of the TPA.IL15IgK in comparison to the native TPA.IL15 in B16.F1 and CT26.CL25 tumor cells. The TPA DNA constructs were transfected into tumor cells, then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL-2. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice and performed in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis(C) Targeted (RGD4C) and non-targeted (NT) TPA particles carrying IL15IgK were produced and used to transduce B16.F1 or CT26.CL25 cells at 0.5x106 or 1x106 TU/cell. Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post-transduction. RGD4C.TPA without IL15IgK (mock) was also included as control. Experiments were repeated twice and shown are the results from a representative experiment. Data are expressed as mean + SEM, one-way ANOVA with Tukey's HSD test was used for data analysis.(D) Evaluation of CTLL-2 proliferation in the presence of media collected from B16.F1 or CT26.CL25 tumor cells after transduction with TPA particles carrying IL15IgK. Cells treated with a recombinant IL2 cytokine were included as a positive control. Data are representative of one experiment, expressed as mean + SEM and shown as change in CTLL-2 number relative to treatment initiation day. Experiments were repeated twice and in triplicates, one-way ANOVA with Tukey's HSD test was used to analyse the data.
Figure 6(A) Tumor-bearing BALB/c mice with established subcutaneous CT26.CL25 tumors were intravenously injected with a single dose, 5x1010 TU/mouse, of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration. Data are representative of one experiment, n = 3, and expressed as mean + SEM. Two-way ANOVA test was used for data analysis.(B) Safety of dose escalation regimens assessed at day 5 following TPA delivery, by evaluating the serum levels of LDH from n = 2 mice/TPA dose of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Serums from untreated mice were also analysed.(C) Representative tumor-bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C.TPA.IL15IgK (RGD4C) or non-targeted (NT) particles. The CT26.CL25 tumor cells stably express a luciferase Luc reporter gene. Untreated mice were also included as controls (CTRL).(D) Changes in tumor viability in mice (n = 4) from day 0 to day 5 post TPA administration. Tumor viability was measured by bioluminescence activity. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Kruskal-Wallis test was used for data analysis.(E) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from all experimental groups.(F) Animal weights (n = 4) were monitored during therapy experiments ad expressed as mean + SEM.
Figure 7(A) The immune profile of CT26.CL25 tumors was investigated using RT-qPCR at day 5 after TPA.IL15IgK administration. A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8+ T cell population; NKp46 for NK cell population. Markers for cytotoxic cell-killing by CD8+ T cells (GZMa, GZMb) and by NK cells (Prf1) were also analysed. Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma (IFN-γ), and FoxP3 for Treg cells. Data shown are representative of n = 5 mice and expressed as mean + SEM. Non-parametric Mann-Whitney test was used for GZMb and TBX2 data analysis, and independent t-test to analyse data of the other markers.(B) TPA particles encoding IL12 were used to treat immunocompetent BALB/c mice bearing subcutaneous CT26.CL25 tumors labelled with a Luc reporter gene. Representative tumor-bearing mice at day 5 post-treatment with targeted RGD4C.TPA.IL12 (RGD4C) or non-targeted TPA.IL12 (NT).(C) Analysis of total flux change in tumors, n = 4 mice, after IL12 immunotherapy. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Mann-Whitney test was used for data analysis.(D) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from two experimental groups.(E) Serial quantification of IL12 in media of B16.F10 cells treated with RGD4C.TPA.IL12 or controls. Data are representative of one experiment, n = 3 biological repeats, and shown as mean + SD Experiments were repeated three times. One-way ANOVA with Tukey's HSD test was used for data analysis.(F) RGD4C.TPA.IL12 immunotherapy of subcutaneous B16.F10 tumors in C57BL/6 mice (n = 4). Data are shown as mean + SEM independent t-test was used for data analysis. Tumor volume changes are shown over time.
Figure 1B. Genome views of RNA read density profiles on representative loci enriched (left) or low (right) in FLAG-HP1γ RNAchIP (IP) relative to input RNA (inp.) in overlaid replicate samples from HP1γ cells stimulated or not with PMA. Orange arrow, position of the locus analyzed in D.C. Genome-wide scatter plot of IP and input normalized (norm.) RNA read counts per gene for the mean of the triplicates; n=21,754; r, Pearson's correlation coefficient between IP and input.D. Relative quantities of RNA in the RNAchIP samples, detected by RT-qPCR with primers aligning on the intron1 of the Fosl1 gene as depicted in B (orange arrow). n=3 independent experiments. Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).E. Heat maps of RNA IP and input signal centered on the summit of RNAchIP peaks of IP versus input detected by MACS2 analysis (10,013 and 2609 peaks) for a representative replicate (replicate 1). Black arrows represent peak summit.F. Percentage of RNAchIP peaks overlapping with indicated chromatin features in MEF samples from ENCODE database (green bars). Overlaps was evaluated by comparison with the list of merged peaks whose genomic location was randomized among genes with one hundred permutations (random, gray bars). Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).
Figure 2B. RNAchIP peaks are enriched in CACACA and GAGAGA motifs. Number of peaks containing at least one exact hexameric motif as indicated, compared to the average number of motifs in peaks whose genomic location was randomized among genes with ten permutations (random, light bars, represent mean and s.d.).C, D. Distribution profiles of average RNAchIP signal in both IP (warm colors) and input (blue colors) over ±2kb centered on intronic (C) and exonic (D) CACACA hexameric motifs oriented in the same orientation (sense) or in opposite orientation (antisense) relative to the overlapping annotated transcript. The antisense CACACA motifs were obtained by querying the transcript sequences with the TGTGTG motif.E. Left, representative example of a locus on the Ppp3ca gene surrounding an RNAchIP merged peak, showing the RNAchIP read density as in Fig 1B. Red and orange arrowheads correspond to oriented CACACA and GAGAGA motifs, respectively. Right, sequence of the RNA surrounding a CACACA motif highlighted in red, as well as a second a neighboring imperfect motif. The sequence was used to design a CACACA-containing RNA oligonucleotide probe (CACACA), compared to a control probe (no-CA) devoid of any related motif.F, Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled RNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.G, Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled dsDNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.H. Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled RNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.