EMBO/SourceData huggingface dataset:
Figure 1B: brain MRI images of II-1 (FLAIR). A: sagittal interemispheric sequence showing profound cerebellar atrophy (arrow); B: transverse sequence showing involvement of the dentate nuclei of cerebellum; C: coronal sequence revealing cortical atrophy, a wide third ventricle, and high signal changes in the thalami, particularly on the left (arrow), D: the thalamic lesion is indicated by an arrow.C: histochemical staining of skeletal muscle from II-2. Combined COX/SDH staining shows scattered COX negative (blue) fibres (arrows). The black bar corresponds to 100 µm.D: respiratory chain complex activities in skeletal muscle. Specific activities (nmol min-1 mg-1) of complex I, II and IV are reduced in II-2 (red) compared to the controls mean (blue); CS activity is also low (<50% of controls) suggesting a reduction of mitochondrial mass (see main text for further details). Each activity was measured in triplicate.E: Western blot analysis of PITRM1 in primary fibroblasts (left) and skeletal muscle (right) of controls (CTR) and subject II-2. Densitometric quantification using the Genetools software is shown below the blots. In blue is PITRM1WT and in red PITRM1R183Q.F: proteolytic activity on two fluorescent oligopeptides (AnaSpec), reported at the bottom of each histogram, by 6-His tagged PITRM1WT and PITRM1R183Q proteins expressed in E. coli and affinity-purified by Ni-agarose chromatography. Values are expressed as arbitrary units per sec per µg of protein (a.u. s-1µg-1). Experiments were performed in duplicate.G: degradation rate of Aβ1-42 by purified 6-His tagged PITRM1WT and PITRM1R183Q proteins. Quantification of these experiments is displayed below the blots. Experiments were performed in duplicate.
Figure 2A: cell growth in glucose (GLU) and galactose (GAL). C29V, C47V, TpLV: immortalized fibroblast cell lines from control individuals; PITRM1V: immortalized fibroblasts from subject II-2, carrying the PITRM1R183Q mutation. pLKO.1: empty vector; sh38: shRNA38; sh41: shRNA41. Each cell line was measured six times. Error bars indicate ±SD. Statistic analysis was by two-way ANOVA post-hoc Bonferroni test ***p<0.001.B: Western-blot analysis of PITRM1 in C29V cells transduced with the empty vector plKO.1 and the two shRNA, sh38 and sh41. SDHB and b-Actin are used as loading controls.C: co-localization of PITRM1 (red) with TOM20 (T20, green) in human fibroblast cells from a control (CTR) and subject II-2. Note that the intensity of PITRM1 immunofluorescence is much lower in II-2 cells than in CTR cells (see main text for further details). Nuclei are stained in blue by DAPI. A white bar corresponds to 10 µM.D: mitochondrial membrane potential (ΔP). In healthy cells with high mitochondrial ΔP, JC-1 forms complexes known as J-aggregates that show an intense red fluorescence. On the contrary in unhealthy cells with low ΔP, JC-1 remains in the monomeric form, showing only green fluorescence. Values are referred to as % of those of the control means, taken as 100%. The results are the mean of 3 independent experiments. Error bars indicate ±SD. Statistic analysis was by two-way ANOVA **p <0.01; ****p<0.0001.
Figure 3A: oxidative growth. W303-1Bcym1Δ strains harbouring the wild-type CYM1 allele (CYM1wt), the cym1R163Q mutant allele or the empty vector were serially diluted from 107 to 104 cells/ml. Five microliters of each dilution were spotted on SC agar plates without uracil, supplemented with 2% glucose, 2% glycerol or 2% ethanol. Plates were incubated at 37°C for 3-7 days.B: oxygen consumption rate (OCR). Cells grown at 37°C SC medium without uracil were supplemented with 0.5% glucose. Values were normalized to the OCR of the CYM1wt strain (49 nmol O2 min−1 mg−1) and represented as the mean of at least three values ± SD. Statistical analysis was by unpaired, two-tail Student's t test: ***p<0.001.C: reduced versus oxidized cytochrome spectra. Peaks at 550, 560 and 602 nm correspond to cytochromes c, b and aa3, respectively. The height of each peak relative to the baseline is an index of cytochrome content.D: respiratory chain complex activities. Biochemical activities of Succinate Quinone DCPIP Reductase, SQDR (CII), NADH-cytochrome c oxidoreductase activity NCCR (CIII), and cytochrome c oxidase (CIV) were measured on a mitochondrial-enriched fraction from cells grown at 37°C as in panel B. Values were normalized to that of CYM1wt strain and represented as the mean of three independent experiments ± SD. Statistical analysis was by unpaired, two-tail Student's t test: **p<0.01; ***p<0.001.E: Western-blot using an anti-HA monoclonal antibody visualizing the CYM1wt and cym1R163Q recombinant proteins both fused in frame with the HA epitope on the C-terminus. Western-blot analysis on total protein extract. Total protein extracts were obtained by strains expressing HA-tagged CYM1wt and cym1R163Q. PGK was used as a loading control and signals were normalized to the wt. The quantification was performed on 5 independent blots.F: prolonged exposure of a Western-blot containing the CYM1wt and cym1R163Q recombinant proteins reveals the presence of a band corresponding to a degradation product in the cym1R163Q lanes (arrow).G: Western blot of Aβ1-42myc monomer and dimer incubated with purified mitochondrial extracts from cells grown at 37°C in SC medium supplemented with 0.15% glucose and 2% galactose. VDAC is used as a loading control. Each experiment was performed in triplicate.
Figure 4A: Western-blot analysis of Pitrm1 protein in brain, muscle and liver of two 4-mo male Pitrm1+/- mice and two Pitrm1+/+ littermates. Densitometric analysis is reported in the histograms below the blots. Pitrm1+/+ is in blue, Pitrm1+/- in red. Error bars indicate ±SDB: representative hindlimb-clasping phenomenon is shown in a 4 mo Pitrm1+/- male mouse, consisting in strong adduction of the hindlimbs when the animal is suspended by the tail; a littermate Pitrm1+/+ control displays the normal reflex, consisting in wide abduction of the limbs. All examined Pitrm1+/- animals displayed this abnormal reflex from 2 months of age.C: rotarod test. Blue and red lines refer to Pitrm1+/+ (n=9) and Pitrm1+/- (n=7) 4 mo animals, respectively. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05; ** p<0.01.D: negative geotaxis test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. * p<0.05.E: cylinder test. The experiment was carried out on the same group of animals as in panel b. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *** p<0.001.F: pole test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. ***p<0.001.
Figure 5A: Western-blot analysis of amyloid-precursor protein (APP) in 6 mo male mice. GAPDH was used as a loading control. Densitometric analysis on a total of 5 independent samples is shown in the histogram on the right.B: morphological analysis of an AD subject and of Pitrm1+/- and Pitrm1+/+ mouse brains. TF: Thioflavin T (thalamus); CR: Congo-red (brain cortex); PL: polarised light (same sections as those stained with CR); Aβ1-42 immunostaining (pons); Ub: ubiquitin immunohistochemistry shown as brownish staining (brain cortex). The white bar indicates 20 µm.
Figure 6A: upper panel: import of Aβ1-42 into mitochondria; lower panel: relative quantification from two independent experiments. Values of Aβ1-42 signal were normalized to HSP60 signal and the resulting value at 5 min without trypsin was arbitrarily chosen as 1. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05.B: upper panel: pulse and chase experiment to assess clearance of Aβ1-42; lower panel: quantification from three independent experiments. Values of Aβ1-42 signal were normalized to HSP60 signal and the pulse value was arbitrarily chosen as 1. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05, **p<0.01.
Figure 1FACS analysis of relative TCRβ+ T cell content in blood (left panel) and tumors (right panel) of mice at the end of treatments as indicated in A. αCD4/αCD8: n=10; IgG: n=6.Tumor burden (left panel), tumor number (middle panel) and average tumor size (right panel) per mouse assessed during colon resection at the end of treatments. αCD4/αCD8: n=10; IgG: n=9.
Figure 2Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative TCRβ+ T cell content in colon (n=10) and tumor (n=22). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Anti-CD3 immunostaining on normal colon (left panel) and a representative colon tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse (right panel: higher magnification of area indicated in middle panel).Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative CD11b+ myeloid cell content in colon (n=16) and tumors (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Anti-Gr1 immunostaining on normal colon (left panel) and a representative colon tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse (right panel: higher magnification of area indicated in middle panel).Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative CD11b+ MHCII- Gr1hi neutrophil (E) and CD11b+ MHCII- Gr1lo monocyte (F) content in colon (n=13) and tumors (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. , Correlation of tumor T cell numbers to sample-matched tumor-derived neutrophil (left panel) and monocyte (right panel) numbers (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.Percentage of T cell subsets among total CD45+ hematopoietic cells in colon (n=10) and tumor (n=11). H, CD4+ TCRβ+ T cells. I, CD8+ TCRβ+ T cells. J, Foxp3+ CD4+ TCRβ+ regulatory T cells (Tregs). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.Percentage of Tregs among total T cells (CD45+ TCRβ+) in normal colon (n=10) or colon tumors (n=11). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.
Figure 3In vitro co-culture of activated T cells with increasing ratios of neutrophils, monocytes or macrophages. T cell proliferation index is numbers of proliferated T cells after three days of indicated co-culture condition relative to the number of proliferated T cells when cultured alone. CD4+ and CD8+ T cells were derived from lymph nodes of wild-type mice. Neutrophils, monocytes and macrophages were derived from colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Each dot represents an individual neutrophil (n=4), monocyte (n=3) or macrophage (n=4) sample. Bars represent mean +/- SD.Tumor neutrophil (left panel) and monocyte (right panel) content after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=10) treatments.Average tumor volume per mouse at the end of αGr1 + CXCR2i (n=9) or IgG + DMSO (n=12) treatments as indicated in B.Anti-CD3 immunostaining on representative tumor sections derived from mice after indicated treatments.FACS analysis of CD45+ TCRβ+ cells, CD45+ TCRβ+ CD8+ cells and CD45+ TCRβ+ CD4+ Foxp3+ cells in extracted tumors after αGr1 + CXCR2i (n=8) or IgG + DMSO (n=7) treatments.FACS analysis of IFNγ (left panel) and IL17A (right panel) expression in tumor extracted T cells after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=7) treatments.
Figure 4Measurement of cytokines present in cell culture supernatants of T cells co-cultured with either tumor-derived neutrophils (T + Ne; n=3) or with tumor-derived monocytes (T + Mo; n=3). Cytokines with a log2 fold change >0.6 or <-0.4 were considered as differentially present in supernatants. Shown is a heatmap of scaled cytokines levels (z-score) differentially present in T + Ne compared to T + Mo. Adjusted p-values were calculated by moderated t-test (limma) and Benjamini post test and were <0.05 only for MMP9 and RELMβ. Supernatants of T cells alone (T only; n=2) served as internal control. T cells cultured with monocytes did not have differential presence of cytokines compared to T cells alone.Co-immunostaining for MMP9 (green) and S100A9 (red) on a representative tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse. Right panel shows a higher magnification of indicated area of the left panel.mRNA expression levels of Mmp9 (C) or Tgfb1, Tgfb2 and Tgfb3 (D) in indicated FACS sorted cell types isolated from mouse colon tumors (n=4). Data are derived from RNA-sequencing and are displayed as reads per kilo base per million mapped reads (rpkm).Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) Protein levels of free active TGFβ1 in cell culture supernatants of indicated conditions determined by ELISA.Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) or with 10µm Galunisertib plus 10µm GM6001 (T + Ne + TGFBRi + MMPi). T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.
Figure 5Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). , Co-immunostaining for pSMAD3 (green) and IGFBP7 (magenta) on representative tumor-containing colon sections derived from Apcfl/fl-Cdx2CreERT2 mice treated with vehicle (A) MMPi (B).Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). Quantification of pSMAD3+ cells (left panel) and pSMAD3 staining intensity per pSMAD3+ cell (right panel) on at least five arbitrarily selected tumor areas per section of MMPi (n=6) or vehicle (n=6) treated mice.effect of in vivo TGFBRi and MMPi treatment of mice for two weeks on colon tumor formation. Apcfl/fl-Cdx2CreERT2 mice were treated with Tamoxifen and, one day post treatment, injected with either MMPi (n=10) or the TGFBRi SB431542 (n=10) at 40mg/kg for two weeks with five injections per week. Vehicle treated mice: n=11. Average tumor size per mouse was assessed during colon resection one week after the end of treatments (E).
Figure 6evaluation of S100A9+ neutrophil and CD8+ T cell infiltration in histologic sections of human CRC tumors. , Anti-S100A9 immunostaining on a representative human CRC section. Arrowheads indicate neutrophil infiltration at the tumor boarder. Co-immunostaining for CD8 (brown) and S100A9 (gray-blue) in the same CRC sample as shown in A.evaluation of S100A9+ neutrophil and CD8+ T cell infiltration in histologic sections of human CRC tumors. Quantification of S100A9+ cells (upper panel) and CD8+ T cells in different areas on CRC tumor sections containing tumor border with adjacent benign mucosa as determined by a specialized pathologist: "Tumor center" was tissue within tumor >200μm from tumor border, "Tumor border" was tissue within tumor <100μm from tumor border and "Adjacent benign mucosa" was mucosa tissue outside tumor <100μm from tumor border. Per section, all identifiable tumor border and adjacent benign areas, as well as three arbitrary tumor center areas were scored. Sections were derived from 10 individual patients.Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. , Neutrophil scores in tumor samples categorized according to CMS subtypes.Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. Comparison of T cell gene expression scores in CMS4 tumors within two publicly available CRC gene expression datasets (median, lower and upper quartiles; horizontal lines define minimum and maximum; dots define outliers). Samples are categorized according to the medians (high = >median; low = neutrophil and TGFβ gene expression scores. Upper panel: TCGA dataset. Lower panel GSE39582 dataset.Proliferation of activated blood T cells in vitro in co-culture with increasing ratios of CRC patient-derived autologous blood neutrophils. CD3+ T cells and CD66b+ neutrophils were isolated and cultured individually for each patient. Bar graphs display relative numbers of proliferated T cells after three days of co-culture. Each dot per condition represents a neutrophil sample from an individual patient (n=13). Bars represent mean + SD.Proliferation of activated blood T cells in vitro in co-culture with autologous blood neutrophils. Cells were derived from both CRC patients (n=2) and healthy volunteers (n=5). T cells and neutrophils were cultured at a 1:1 ratio and treated with vehicle (T + Ne) or 10µm Galunisertib (T + Ne + TGFBRi). Each line represents a neutrophil sample from an individual healthy volunteer.
Figure 2Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of Dieldrin for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of PMA for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of either Dieldrin or PMA for the indicated times. Abundance of mRNA for the indicated genes by RT-qPCR.Jurkat cells were exposed to either DMSO, PMA in DMSO, or dieldrin in DMSO for 30 min in triplicates. RNA-seq was then performed on cDNA libraries prepared with poly(A) selection: principal component analysis on the 485 genes the most affected by DMSO, PMA, or Dieldrin.
Figure 3Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing an example of a HERV becoming a site of divergent transcription upon exposure of the Jurkat cells to dieldrin. Top tracks show coverage from sequencing of poly(A)-enriched libraries for comparison. All coverage tracks are at the same scale and all the sequencing result files are of comparable size. Blue and red reads (rds) are in opposite orientation. CAGE peaks use the same color code. Reads mapping inside a series of 4686 HERVs located away (30Kb) from protein-coding genes were quantified both in the poly(A)-enriched and the ribo-depleted data. Read counts from each replicate were then summed and plotted.Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Distribution of regions annotated "LTR" in RepeatMasker relative to sites of divergent transcription in Jurkat cells treated with either DMSO or dieldrin in DMSO.Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Jurkat T cells were treated with either DMSO (vehicle) or 100μM dieldrin for the indicated times. Abundance of IL2 mRNA was assessed by RT-qPCR. Data shown are means +/- SEM from four independent experiments. Significance (p-value) was estimated using the two-sided student t test appropriate for small sample numbers. ***: p-value<0.001.Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing increased divergent transcription inside a HERV sequence upon exposure of the Jurkat cells to dieldrin in the intergenic region between MUC21 and MUC22. Color code as in (A).
Figure 4A human phospho-kinase antibody array was probed with extracts from Jurkat cells treated with either DMSO, dieldrin, or PMA for 30 min as indicated. The array processed as a western and revealed by ECL. Graphic shows selected antibodies. Values shown are means +/- SEM from two measures.Jurkat cells were treated with either DMSO (vehicle), dieldrin, or PMA for the indicated times. Cell extracts were resolved by PAGE and western blotting was carried out with the indicated antibodies. Nearest size marker is indicated to the right (kDa).Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.Jurkat cells treated as in (C) were subject to fractionation as indicated in the Material and Method section to distinguish a fraction with mostly RNAPII S2p (F1), one with mostly histone H3 (F3), and one with both RNAPII S2p and histone H3 (P). Fractions were resolved by PAGE, and western blots were carried out with the indicated antibodies.
Figure 2(A) Anti-acetyllysine Western blot analyses and total protein stains of E. coli cell extracts. Cellular protein extracts before and after induction of His6-MBP-GNAT expression were separated on 12 % acrylamide gels and immunoblotted by using an anti-acetyllysine antibody or stained with Coomassie Dye. GNAT1, 2, 5, 6, 7 and 10 as well as His6-MBP control were expressed in E. coli BL21(DE3)pLysS. GNAT3 and 4 were expressed in Rosetta(DE3). In the total protein stains, recombinant GNAT protein constructs were highlighted by blue arrowheads. Protein expression before (ni) and after (i) induction with IPTG is indicated. The luminescence signal, indicating acetylated proteins, was usually recorded after 40-120 seconds. Since MBP-GNAT4 expression resulted in a saturated signal, the luminescence was additionally recorded for 10 seconds (indicated by an asterisk).
Figure 3(A) Number of characterized substrates with significant increases of NTA yields. The NTA yields of all retrieved N-termini upon a given GNAT expression was divided into four categories and each column is the result of the sum of all categories: 50-100%, dark green; 20-50%, light green; 10-20%, orange; 5-10%, red. The number of protein occurrence with a yield higher than 30% is also displayed to visualize the most significant acetylation status of each GNAT. The respective overexpression level of each GNAT as assessed from gel electrophoresis (++++ is for very high expression, +++ for high, ++ for medium, + for intermediate).