EMBO/SourceData huggingface dataset:
Figure 6Representative Western blot showing levels of mitochondrial proteins, as indicated by the presence of aconitase, VDAC, and Tom20, in the total lysates of PC12 cells with Q23 or Q74 in an in vitro reconstituted micro-mitophagic system. NT: no inactive GAPDH treatment; G: inactive GAPDH treatment. n = 3. *P = 0.0007; **P = 0.001; #P = 0.008.Representative Western blots showing levels of mitochondrial proteins (aconitase and Tom20) in the total lysates of fibroblast cells (juvenile and adult) and mice striatal cells with Q7 or Q111 in an in vitro reconstituted mitophagic system. n = 2. §P = 0.01; §§P = 0.02; *P = 0.04; **P = 0.02; #P = 0.001; ##P = 0.024.Representative Western blot showing levels of mitochondrial proteins (aconitase and Tom20) in the total lysates of PC12 cells. The cells were treated with 1 mM of 3-methyladenine (3MA) for 5 h prior to the micro-mitophagy reconstitution assay. n = 2. *P = 0.001; **P = 0.01; *#P = 0.003; **#P = 0.01; #P = 0.02; §P = 0.049; #§P = 0.004; ##§P = 0.007.Western blot showing the level of Atg5 expression in the Atg5+/+ (WT) and Atg5−/− (KO) MEF cells.Western blot showing levels of GAPDH association with mitochondria in Atg5 KO MEF cells. n = 2. *P = 0.04.Western blot showing relative mitochondrial mass in the total lysates of Atg5 KO MEF cells. n = 2. **P = 0.03; ***P = 0.02. Expression levels of Q23 or Q73 were detected with anti-His antibody.Western blot showing relative mitochondrial mass in the total lysates of Atg5 KO MEF cells without and with recombinant inactive GAPDH treatment (represented as NT and G, respectively). n = 2. *P = 0.02; **P = 0.03; *#P = 0.03; #P = 0.005.
Figure 1A Left, kymograph from sequential epifluorescence (20×) images showing cytosolic Ca2+ oscillations in speed‐fluctuating immature DC after slow motility phases (upper and lower panel) and a change in direction (lower panel). DCs were loaded with the Ca2+ dye Oregon GreenBAPTA 1‐AM, introduced in micro‐channels, and imaged every 10 s. Right, sequential epifluorescence (20×) images (1 image/20 s is shown). Instantaneous velocities and intracellular calcium oscillations were measured as described in Materials and Methods. Scale bar: 10 μm.B, C Mean cell velocity of immature DCs (n > 125 cells from two independent experiments) (B) or CD8+T lymphocytes (n > 40 cells from one experiment) (C) migrating in micro‐channels with or without BAPTA. Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.D Mean intracellular calcium concentration in speed‐fluctuating (ΔV/V0 > 0.3; ΔV/V0 relative speed variation divided by the global median velocity) and non‐speed‐fluctuating (ΔV/V0 0.3) immature DCs (n = 187 cells from more than three independent experiments). P‐values were calculated using a Mann-Whitney test.E, F Velocity fluctuations (ΔV/V0) (E) or mean Ca2+ activities (F) displayed by DCs migrating in micro‐channels in the presence of 100 ng/ml CCL3. P‐values were calculated using a Mann-Whitney test, with respect to non‐treated DCs (NT).G Mean Ca2+ activities during phases of fast (> 50% of max velocity) and slow ( 50% of max velocity) motility in speed‐fluctuating DCs (Supplementary Fig S2F) (n = 64 cells, from three independent experiments). Activities were normalized to the mean Ca2+ activity shown by each cell during the entire movie. The P‐value was calculated using a Mann-Whitney test.
Figure 2A Relative gene expression of IP3R1 (blue), IP3R2 (red), and IP3R3 (green) in IP3R‐silenced DCs. DCs were infected with two lentiviruses encoding for different shRNAs for IP3R isoforms. ShScramble‐infected DCs were used as a control. Gene expression was determined by a quantitative PCR. It was calculated with respect to GAPDH expression and normalized to the levels observed in the shScramble. The median plus standard deviation of three independent experiments are shown.B Immunoblot for IP3R type 1 and IP3R type 3 in IP3R‐silenced DCs. Actin was used as a control. IP3R3 appears as two bands as previously described (22).C Immunoblot for IP3R type 1 (top) and IP3R type 3 (bottom) in DCs transduced with lentivirus encoding for different IP3R shRNAs. Actin was used as a control.D Relative IP3R2 expression in ShScramble‐infected DCs (gray), shIP3R1 (blue), and IP3R3 (green)‐silenced DCs. The experiment was performed as described in (A).F Transmigration assay of shScramble‐, IP3R(1,3)A (blue)‐, IP3R(2,3)B (red)‐, and IP3R(1,3)C (green)‐expressing DCs. Cells were loaded in the upper chamber of a 5‐μm pore collagen‐coated transwell assay, recovered from the upper and lower chambers after 16 h, and counted. The median from three independent experiments is shown.G Quantification of the mean cell velocity of shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐silenced immature DCs migrating in micro‐channels. shScramble‐infected DCs were used as a control (gray) (n > 100 cells from three independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.H Quantification of the mean cell velocity of shIP3R(1,3)C (green)‐silenced immature DCs migrating in micro‐channels in the presence of 2 mM BAPTA. shScramble‐infected DCs were used as a control (gray) (n > 70 cells from two independent experiments). P‐values were calculated using a Kruskal-Wallis test.
Figure 3Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).A Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels. Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).B Dot plot showing velocity fluctuations and migration speeds of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs.Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).C Inverted sequential epifluorescence images (20×) of a representative control DC (left) and an immature shIP3R(1,3)C‐silenced DC (right) migrating in micro‐channels. DCs were differentiated from LifeAct‐GFP transgenic mice and imaged every 2.2 min.Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).D Percentage of DCs changing their direction of locomotion while migrating in micro‐channels. P‐values were calculated with a paired test with respect to the shScramble condition.Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).E Dot plot showing the frequency of changes in direction of immature DCs while migrating in micro‐channels. Bars show the mean plus the standard deviation. A Kruskal-Wallis test was applied for statistical analysis.
Figure 4Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.A Graph showing the DC mean instantaneous speed. P‐values were calculated with a t‐test analysis.B Mean instantaneous speed distribution of migrating DCs.Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.C Representation of individual DC trajectories.Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.D Path persistence (calculated as the ratio between the effective length and the total length of the cell trajectory) of migrating DCs. P‐values were determined with a t‐test analysis.Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.E Bars showing the proportion of the effective length displacement (L) of DCs. A Fisher's exact test (P‐value 2.2 × 10−16) was used for statistics.
Figure 5A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs transduced with the shScramble or shRNAs for IP3Rs (shIP3R(1,3)A′, shIP3R(2,3)B′, and shIP3R(1,3)C′). Actin was used as a control.B Inverted sequential epifluorescence images (20×) showing myosin IIA‐GFP localization of a representative shScramble (left)‐ and shIP3R(1,3)C‐expressing DC (right) migrating in micro‐channels. Myosin IIA‐GFP knock‐in DCs were imaged every 1 min (1 out of 2 images is shown). Asterisks highlight changes of direction during locomotion.C, D Myosin IIA‐GFP localization (red) versus velocity fluctuations (blue, ΔV/V0) in a representative shScramble‐transduced DC (C) or shIP3R(1,3)C‐silenced (D) migrating in micro‐channels. DCs were recorded as in (B).E Quantification of the interval between curves (highlighted in D) obtained for myosin IIA positioning and velocity from shScramble‐ and shIP3R(1,3)C‐expressing DCs. Cells were recorded as in (B) (n = 20 cells from two experiments). The Mann-Whitney test was applied for statistical analysis.
Figure 6A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor, ML7.B, C Analysis of immature DCs migrating in micro‐channels in the presence of ML7 or DMSO as a control (n > 170 cells from two independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (B). Whiskers show 10-90 percentiles plus the median. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (C) in direction of DCs migrating in micro‐channels. Mean plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.D, E Analysis of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels in the presence of 20 μM ML7 (n > 70 cells per condition from 3 independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (D). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (E) in direction observed in DCs migrating in micro‐channels. Means plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.
Figure 1Quantification by western-blot analysis of EZH2 and H3K27me3 levels at each stage of de novo transformation. UT: untransformed, PN: pre-neoplastic, TR: transformed. Histone H3 is used as a loading control. The graph on the right displays the H3 normalized densitometric values of western-blot band from untreated and DMSO treated cells. Values represent mean ± SEM from three biological replicates. One asterisk indicates p-value < 0.05 (one-tailed unpaired Student's t-test). ns: non-significant.Volcano plot showing the relative enrichment of EZH2 binding at all sites detected in either untransformed or transformed cells. Numbers within the volcano plot indicate differential binding sites with a p-value ≤1e-20 and a fold-change in normalised tag count ≥1.5. Enrichment significance calculated based on a Poisson distribution using the 'getDifferentialPeaks' function in homer (see: methods). P-values of > 1e-100 were set to 1e-100 for display reasons. Tracks on the right show representative high- and low-magnitude EZH2 differential peaks. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. Blue bars represent regions called as an EZH2 binding site. UT: untransformed, TR: transformed.
Figure 2Western-blot analysis of H3K27me3 levels after treatment of cells with an EZH2 inhibitor (EZH2i) or DMSO as control for 12 days. Histone H3 is used as a loading control. Values represent H3-normalized densitometric values of the H3K27me3 bands, and are expressed relative to the relevant DMSO control. UT: untransformed, PN: pre-neoplastic, TR: transformed.Quantification of differentially expressed genes (false discovery rate (FDR) ≤ 0.01, Log2FC ≥ 1/≤-1 and maximal transcripts per million (maxTPM) ≥ 1) detected by RNA-seq in each cellular state upon EZH2i treatment. UT: untransformed, PN: pre-neoplastic, TR: transformed.Heatmap showing the relative expression of genes identified by the multi-step filtering strategy in the indicated cellular states. UT: untransformed, PN: pre-neoplastic, TR: transformed. Colours represent log2-transformed, row-normalised TPM values.
Figure 4Expression of EMX2 in normal brain and GBM patient samples. Data sourced from the Repository of Molecular Brain Neoplasia Data (REMBRANDT). Four asterisks indicate p-value < 0.0001 (two-tailed Mann Whitney U test). Bars represent median ± interquartile range. Values are expressed relative to the mean of normal brain samples. N: 21 for normal brain, 214 for GBM. Note that the extent of EMX2 repression in GBM patients is likely underestimated due to the possible presence of normal adjacent tissue in the analysed samples, and due to the intrinsic background noise of microarrays, which limits detection of truly silenced genes.Relative expression of HOX genes in normal brain and GBM patient samples as detected by microarray analysis. Data sourced from REMBRANDT. Values are expressed relative to the median of normal brain samples. In the boxplots, the top, middle and bottom box delimiters represent the 75th, 50th and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile - 1.5*interquartile range, respectively. One asterisk indicates p-value <0.05 (two-tailed Mann Whitney U test corrected for multiple comparison using Holm's method). N: 21 for normal brain, 214 for GBM. Data was not available for HOXD8.Covariance between EMX2 and EZH2 expression levels in GBM patient samples, as detected by microarray analysis. Data sourced from REMBRANDT. P-value and correlation coefficient (r) of the covariance are shown (Spearman rank correlation). Every dot is a patient. N: 214.Expression levels of EMX2 (top) and EZH2 (bottom) in tumour or adjacent normal regions laser microdissected from human GBM tumours as detected by RNA-seq. Data sourced from the Ivy Glioblastoma Atlas. FPKM: fragments per kilobase of transcript per million. The significance of the differential expression in normal and tumour regions across patients is indicated (two-way ANOVA). Bars represent mean ± SEM. N: 3 regions sampled for each GBM tumour, 2, 3, 2, 3, 2, 3, 3 and 1 regions for normal tissue of patients 1-8, respectively.Hematoxylin and eosin (H&E) staining of a human GBM tumour. The black square represents the approximate location of the field shown in G (left image). Serial sections were used for H&E and RNA-FISH. Scale bar: 300 µm.Visualisation of EZH2 (green) and EMX2 mRNA (red) in a human GBM tumour by RNA FISH. Nuclei were counterstained with DAPI. Scale bar: 20 µm.Expression of EMX2 (left) and EZH2 (right) in glioma patient samples, grouped according to tumour grade, as detected by RNA-seq. Data sourced from the Chinese Glioma Genome Atlas. Four asterisks indicate p-value < 0.0001 (Kruskal-Wallis test). Bars represent median ± interquartile range. N: 109 for grade II -, 72 for grade III, 144 for grade IV. RPKM: Reads per kilobase of transcript per million.
Figure 5Proliferation assay examining the effect of EMX2 ectopic expression in U-87 MG and DBTRG-05MG GBM cells, RFP is used as a control. Values represent mean ± SEM from three biological replicates. The fold change in cell number after eight days of proliferation whilst expressing the exogenous protein is shown relative to an uninduced control. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). Similar results were reported in [103] using other GBM cell lines.Transplantation assay comparing the growth kinetics of subcutaneous DBTRG-05MG-induced tumours expressing EMX2 at levels comparable to those expressed in normal cells, or expressing RFP as a control. Values represent mean ± SEM from six tumours. Three asterisks indicate p-value < 0.001 (two-tailed single sample Student's t-test). Similar results were obtained in an independent experiment using a distinct batch of transduced cells.MRI scans showing representative brain tumours six weeks post-injection of DBTRG-05MG cells expressing EMX2 or RFP. Axial anatomical scan and coronal signal intensity map indicate location of tumour in RFP control (yellow arrow). Contrast enhancement in post-contrast images indicates tumour blood brain barrier breakdown. Signal was not present in pre-contrast images. Images show the same MRI slice position between mice. Scale bar: 2mm. Quantification of brain tumour size in mice injected with EMX2- or RFP- expressing DBTRG-05MG cells. Values represent mean ± SEM from four control and three EMX2 tumours. Five mice per conditions were injected but three (one for RFP, and two for EMX2) had to be excluded from the study due to complications from the procedure.
Figure 1Representative images of euploid control or ArCK cells interacting with NK cells. The NK cell-mediated killing was measured at a 2.5:1 (NK cells: target cells) ratio and was recorded by live cell imaging for 36 hours at a 30-min interval. TO-PRO-3 (1 μM) was added to the medium at the same time of NK cell addition to measure cell membrane integrity. Phase contrast (top) and TO-PRO-3 signal (bottom) from the same field were presented. Arrowheads indicate ArCK cell death. All images were acquired at the same exposure time and light intensity. Scale bar 20 μM.NK cell-mediated cytotoxicity across various NK cell to target cell ratios. Either euploid control (Ctrl) or ArCK cells were co-cultured with NK cells at the indicated NK cell: target cell ratios. The cumulative killing of target cells was measured. n=3 biological replicates; mean± SEM. p< 0.0001 for all four NK cell: target cell ratios, KS test.
Figure 2NK cell-mediated killing for doxorubicin (Doxo), palbociclib (Palbo), and nutlin3 (Nutlin) treated samples (NK cell: target cell =2.5:1). n=3 biological replicates; mean± SEM. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p< 0.0001; Ctrl vs. Nutlin, p< 0.0001; KS test.NK cell-mediated cytotoxicity towards torin1-treated cells. Torin1 treated (Torin) cells were generated as described in (F) and the NK cell killing assay was performed as described in Figure 1. n=3 biological replicates; mean± SEM. Ctrl vs. Torin, p= 0.79, not significant (n.s.); KS test.
Figure 3   γ-H2AX foci were analyzed in ArCK and G1 arrested cells (generated as described in Figure 2). At least 50 cells were analyzed per condition per replicate. n=2 biological replicates; individual values and mean are shown. The distribution of the foci number in each treated condition was compared to that of control using Kolmogorov-Smirnov test. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p= 0.07, n.s.; Ctrl vs. Nutlin, p= 0.11, n.s.; Ctrl vs. ArCK, p= 0.0007.   Secreted cytokine and interferon levels were determined in cell supernatants. Media were collected after 36 hours of incubation with cells grown as described in Figure 1 and 2. Cytokine and interferon levels were shown as fold change normalized to euploid control cells. Individual values and mean are shown; n=2 biological replicates.NK cell medium was incubated with either euploid control or ArCK cells for 12 hours. At the time of NK cell addition, media were switched between ArCK and euploid control cells (Ctrl). NK cell killing was measured as described in Figure 1C. For reference, NK cell killing of ArCK and euploid control cells (Ctrl) without medium switch were performed side by side and plotted on the graph. Black, euploid control cells without medium switch; red, ArCK cells without medium switch; blue, euploid control cells in ArCK cell condition medium; green, ArCK cells in euploid control cell condition medium. n=3 biological replicates; mean± SEM. Ctrl vs. ArCK, p< 0.0001; Ctrl vs. Ctrl in ArCK med, p< 0.0001; Ctrl vs. ArCK in Ctrl med, p< 0.0001; ArCK vs. ArCK in Ctrl med, p= 0.0002; KS test.
Figure 4RT-qPCR quantifying NF-κB downstream target gene expression in ArCK and euploid control (Ctrl) cells. n=6 biological replicates; mean± SEM. IL-1b, *p= 0.029; IL-6, *p= 0.032; IL-8, **p= 0.008; unpaired t-test.
Figure 5ArCK or euploid proliferating control cells were treated with either DMSO or the NF-κB inhibitor BMS-345541 (5 μM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean± SEM. ArCK vs ArCK NF-κB inhibitor, p< 0.0001; KS test. (H, I) The effect of inactivating both RELA and RELB on NK cell-mediated cytotoxicity in 7-day doxorubicin (H) and nutlin3-treated (I) cells. n2 biological replicates; Doxo-c1 vs. Doxo RELA RELB-c1, p = 0.02. Nutlin-c1 vs. Nutlin RELA RELB-c1, p = 0.44, n.s.; KS test.
Figure 6HCT116 (left) or DLD1 cells (right) were treated with either DMSO or the Mps1 inhibitor reversine (500 nM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean±SEM. HCT116 vs. HCT116 Mps1 inhibitor, p= 0.44, n.s.; DLD1 vs. DLD1 Mps1 inhibitor, p= 0.44, n.s.; KS test.