EMBO/soda-nel huggingface dataset:
Loss of TMEM106B potentiates lysosomal and FTD-like pathology in progranulin deficient mice (2020)
Figure 2

Figure 2. Expression analysis reveals global dysregulation of gene clusters associated with autophagy and microglial activation in double knockout mice.

A-C. Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed brain mRNAs from Tmem106b-/- and wild type mice. From 680 detected genes 21 are significantly upregulated, while 10 are significantly reduced. (B) Comparison of Grn-/- and wild type mice. From 680 detected genes 12 are significantly upregulated, while 6 are significantly reduced. (C) Volcano plot for Grn-/-/Tmem106b-/- and wild type mice. From 680 detected genes 79 are significantly upregulated, while 24 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 9 genes are significantly altered in all 3 genotypes in comparison to the wild type. 7 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 15 significantly altered genes. Tmem106b-/- and Grn-/- mice share one significant altered gene. Data represent the mean ± SD.

E. Gene set analysis based on NanoString advanced analysis R-script included in the Neuropathology panel.

Data information: For statistical analysis of volcano plot data, unpaired two-tailed Student's t-test was performed between individual genotypes and the wild type control condition. Significance was accepted when p < 0.05 as indicated by the dotted line. Source data including exact values are available online for this figure.
Loss of TMEM106B potentiates lysosomal and FTD-like pathology in progranulin deficient mice (2020)
Figure 3

Figure 3. Expression analysis reveals enhanced dysregulation of genes associated with microglia and astrocyte function.

A-C. Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type, 4 Grn-/-, 4 Tmem106b-/-, 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. (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. (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. (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.

E. Gene set analysis based on NanoString advanced analysis R-script included in the Neuroinflammation panel.

Data information: For statistical analysis of volcano plot data, unpaired two-tailed Student's t-test was performed between individual genotypes and the wild type control condition. Significance was accepted when p < 0.05 as indicated by the dotted line. Source data including exact values are available online for this figure.
Loss of TMEM106B potentiates lysosomal and FTD-like pathology in progranulin deficient mice (2020)
Figure 4

Figure 4. Astrogliosis and microgliosis in different brain regions of Grn-/-/Tmem106b-/- mice.

Western blot analysis of GFAP and IBA1 in total brain lysates from 4.5-month-old mice with the given genotype. (n = 3 biological replicates per 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). Scale bar indicates 100 µm.

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.

Data information: (B), one-way ANOVA with Tukey's post hoc test was used to compare individual genotypes. Significance is indicated; ***p < 0.001; and ****p < 0.0001.

Source data including exact p‐values and quantification values are available online for this figure.
Loss of TMEM106B potentiates lysosomal and FTD-like pathology in progranulin deficient mice (2020)
Figure 5

Figure 5. Enhanced expression of proteins associated with activated microglia in Grn-/-/Tmem106b-/- mice.

Western 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. Data represent the mean ± SD.

Expression of CD68 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (TH = Thalamus). Scale bar indicates 25 µm.

Expression of CLEC7A and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Scale bar indicates 25 µm.

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.

Data information: For statistical analysis of protein levels (B), one-way ANOVA with Tukey's post hoc test was used to compare individual genotypes. Significance is indicated; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Source data including exact p‐values and n numbers are available online for this figure.
Loss of TMEM106B potentiates lysosomal and FTD-like pathology in progranulin deficient mice (2020)
Figure 6

Figure 6. Elevated lysosomal enzyme levels and activity in Grn-/-/Tmem106b-/- mice.

Western 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). Data represent the mean ± SD.

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).

Data information: For statistical analysis of normalized data (B, C), one-way ANOVA with Tukey's post hoc test was used to compare individual genotypes. Significance is indicated; ns p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Source data including exact p‐values and n numbers are available online for this figure.
Loss of TMEM106B potentiates lysosomal and FTD-like pathology in progranulin deficient mice (2020)
Figure 7

Figure 7. Widespread impairment of autophagy and protein degradation in Grn-/-/Tmem106b-/- mice.

Immunofluorescence 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.

Data information: For statistical analysis of normalized data (C), one-way ANOVA with Tukey's post hoc test was used to compare individual genotypes. Significance is indicated; ns p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Source data including exact p‐values and n numbers are available online for this figure.
Loss of TMEM106B potentiates lysosomal and FTD-like pathology in progranulin deficient mice (2020)
Figure 8

Figure 8. Enhanced TDP-43 aggregation and phosphorylation in Grn-/-/Tmem106b-/- mice.

Immunofluorescence 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. (n = 3 biological replicates per genotype).

Quantification of (C) Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.

Data information: For statistical analysis of normalized data (D), one-way ANOVA with Tukey's post hoc test was used to compare individual genotypes. Significance is indicated; ns p > 0.05; *p < 0.05; **p < 0.01.

Expanded View Figure Legends
USP14 inhibition corrects an in vivo model of impaired mitophagy (2018)
Figure 1

Figure 1. USP14 inhibitor IU1 treatment induces mitophagy in SH-SY5Y cells.

A Western blot analysis of LC3 in SH-SY5Y cells after IU1 treatment (100 μM, 24h). Graphs represent mean± SEM. N= 8.

B-D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control (DMSO) or IU1 treated cells. At least 55 cells were analysed from 3 different experiments. Graphs represent mean± SEM.

E Western blot analysis of mitochondrial marker proteins ATP5a, HSP60 and normalised by actin after 24h of DMSO (C) or IU1 (100 μM) treatment. N=3. Graphs represent mean± SEM.

F-I Confocal images of cells transfected with mito-YFP and treated with DMSO (F, control) or IU1 (G) for 24h. Fragmentation index (H) and total mitochondrial volume / cell were measured and represented in (I), mean ±SEM. At least 35 cells were analysed from 3 different experimental conditions. Scale bar 10 μm.

J-L Mito-YFP transfected cells were treated with IU1 (100 μM, 24h), fixed and immuno labelled for LC3. Number of LC3 dots / cell (K) and LC3 dot overlapping mitochondria (L) were counted. At least 35 cells from 3 independent experiments were analysed. Scale bar: 10 μm.

M-N Cells were transfected with mito-keima and treated with DMSO or IU1 for 24h and subjected to FACS analysis, counting 10,000 cells per experiment. (M) represents the shift of the signal intensity in IU1 treated cells when excited at 560 nm (false colour). (N) represents the average intensity of the mito-keima protein when excited at 560 nm and the ratio between 560: 405 nm. Graphs represent mean ± SEM from 3 different sets of experiments.

Student's t test. *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001.
USP14 inhibition corrects an in vivo model of impaired mitophagy (2018)
Figure 2

Figure 2. IU1 facilitated mitochondrial clearance is DRP1 and Mfn2 dependent.

A-B Western blot analysis of the indicated proteins in cell lysates from SH-SY5Y cells, treated with different concentrations of IU1 (1-100 μM) for 24h. Bar graphs represents mean ± SEM. Blots are representative of three independent experiments. ANOVA followed by Dunnets test.

C-D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito-YFP (C) with / without IU1 treatment (100 μM, 48h). Scale bar 10 μm. (D) Mitochondrial volume / cell was measured as described and represented as mean ±SEM. At least 35 cells were evaluated.

E-F Western blot analysis of HSP60 and ATP5a, as mitochondrial marker in the indicated knock out MEF cells (with / without the indicated DRP1 variant) after 48h of IU1 treatment. Bar graphs represent mean ± SEM, n= at least 3. Student's t test;

*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
USP14 inhibition corrects an in vivo model of impaired mitophagy (2018)
Figure 3

Figure 3. IU1 treatment mediated mitophagy is Parkin / PINK1 independent.

A-D Wild type MEF (A), Hela (B) and PINK1 KO MEF (C) cells were treated with IU1 (48h, 100 μM) and electron microscope images were evaluated for autophagic vesicle formation. (D) bar graphs represents mean± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments; at least 40 cells were analysed from each group.

E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type. Bar graphs represent mean ± SEM. N= at least 3 independent experiments. Student's t test.

*P ≤ 0.05, **P ≤ 0.01.
USP14 inhibition corrects an in vivo model of impaired mitophagy (2018)
Figure 4

Figure 4. IU1 treatment mediates the translocation of the 20S proteasome subunit to mitochondria.

A-B SH-SY5Y cells were immunostained for ATP5a (green) and 20S proteasome core (red) after IU1 treatment (6h-24h). Scale bar 10 μm. Colour mapping of the co-localisation of HSP60 and 20S proteasome from the selected regions were generated by image j and represented in the bottom panel. Intensity of the co-localisation is represented in the colour map scale at the bottom of the panel. (B) 20S proteasome subunit and mitochondria co-localisation was quantified by measuring Mander's coefficient. Bar graphs represent mean fold change (mean ± SEM) from 3 different experiments. At least 30 cells were evaluated for the calculations. ANOVA followed by Dunnet's test.

C Electron microscopy images showing subcellular localization of immuno- gold labelled 20S subunit of proteasome complex after 12h or 24h of IU1 treatment. Experiments were repeated twice in two biological replicates with similar results. The contrast in the magnified region is enhanced from the original image to highlight the immunogold labelled signals.

D-E Protein proximity ligation assay for Prohibitin2 (PHB2) and LC3 after 24h IU1 treatment. Images showing signal are merged with bright field images to count the signal dots (cyan false colour) as depicted in the figure. In the inset only fluorescence signal is shown, without merging with the bright field from the selected regions. Scale bar 10 μm. (E) Bar graphs represent mean ±SEM of the number of signal dots per cell from 3 different experiments. Student's t test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
USP14 inhibition corrects an in vivo model of impaired mitophagy (2018)
Figure 5

Figure 5. USP14 inhibition or knock down ruptured mitochondrial membrane integrity.

A, C, E, G, I Electron microscope images of mitochondria from SH-SY5Y, MEF WT, MEF PINK1 KO Hela and Drosophila thoracic muscle, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted. For flies, more than 200 mitochondria from each group were counted from 5 different experiments.

B, D, F, H, J Representative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experiments.

Student's t test; *P ≤ 0.05, **P ≤ 0.01.
USP14 inhibition corrects an in vivo model of impaired mitophagy (2018)
Figure 6

Figure 6. USP14 knockdown corrects life span, locomotor impairment, mitochondria respiratory defects and muscle degeneration of PINK1-deficient flies

A Life span analysis of flies of the indicated genetic background. At least 50 flies were monitored from each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001).

B Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. n=4-7. ANOVA followed by Newman Keuls test.

C Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows respiratory control rate (RCR) calculated as described in materials and methods. Graphs represent mean± SEM. N=4-5, ANOVA followed by Newman Keuls test.

D Dopamine content in the fly heads (15 day old) was measured by HPLC and the individual fold change values are represented in the scatter graph. Graphs represent mean± SEM. N= 14, 11 and 11 fly heads for control, PINK1 and PINK1+USP14 KD fly respectively. ANOVA followed by Newman Keuls test.

E Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated 3 times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and depolarised mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes.

*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
H2A.B is a cancer/testis factor involved in activation of ribosome biogenesis in Hodgkin Lymphoma (2021)
Figure 1

Figure 1. H2A.B is upregulated in all types of primary Hodgkin Lymphoma.

A-F Immunostaining of primary HL with H2A.B (brown, indicated by black arrows) with a nuclear counterstain (blue). Human testis (positive control) (A), Human tonsils (negative control) (B), Nodular sclerosis classical cHL, double staining with CD15 (pink) to identify HRS cells and H2A.B (brown) (C), Mixed cellularity cHL (D), Lymphocyte rich cHL (E), Nodular sclerosis HL (F). Scale bar for panels A, B, D-F is 50 µm; scale bar for panel C is 15 µm.

G Percentage of H2A.B-positive HRS cells in all 81 tumours. Lymphocyte Rich cHL (LRCHL), Mixed Cellularity cHL (MCCHL), Nodular sclerosis cHL (NSCHL), Nodular Lymphocyte Predominant HL (NLPHL), unclassified cases (U/C). Error bars are standard deviations, and the black line represents the average.
H2A.B is a cancer/testis factor involved in activation of ribosome biogenesis in Hodgkin Lymphoma (2021)
Figure 2

Figure 2. H2A.B is upregulated in Hodgkin Lymphoma cell lines.

A Relative mRNA expression levels of H2A.B in L1236, L428, HDLM2 HL cell lines and Germinal centre B lymphocytes (GC) compared to the Ramos B lymphocyte cell line. The results are presented as the mean ± SD of three biological replicates.

B Western blot analysis of H2A.B protein expression relative to histone H3 protein expression. Error bars represent the standard deviation for three biological replicates, and the red line shows the average.

C Subcellular fractionation of L1236 and L428 cells using KCl salt. CN, cytoplasmic and nucleoplasmic fraction, LB, Loosely bound to chromatin, CHR, chromatin fraction. Note that the H3 Western blot signal in CHR is overexposed.

Source data are available online for this figure.
H2A.B is a cancer/testis factor involved in activation of ribosome biogenesis in Hodgkin Lymphoma (2021)
Figure 3

Figure 3. H2A.B is located at rDNA repeats.

A H2A.B (red) is co-localized with fibrillarin (a nucleolar marker, green) in the nucleoli of L1236 cells. DAPI (blue) was used to counterstain nuclei. Scale bar is 10 µm.

B Primary tumours stained with anti-H2A.B (red) and anti-fibrillarin (green) antibodies. Co-localization appears as a yellow signal, and DAPI (blue) counterstains nuclei. NSCHL, nodular sclerosis classical cHL; MCCHL, mixed cellularity cHL, scale bar is 25µm.

C ChIP-qPCR showing the enrichment of H2A.B-containing nucleosomes at rDNA chromatin. ChIP with anti-H2A.B antibody was performed in L1236 and L428 cells followed by qPCR with rDNA primers corresponding to untranslated and spliced-out regions. Intergenic and enhancer regions were used as negative controls. The results are presented as the mean ± SD of three biological replicates. Below the graph is a schematic diagram of an rDNA repeat. PRMTR, promoter; ETS and ITS, external and internal transcribed regions, respectively; IGS, intergenic spacer; ENHCR, enhancer region. Enrichment was calculated relative to input DNA. Red lines show the location of the amplification regions (PRMTR, 5' ETS, ITS2, 28S, IGS and ENHCR).

D H2A.B interacts with Pol I. Western blot showing co-immunoprecipitation of RPA194 with H2A.B. The Pol I subunit RPA194 was immunoprecipitated from L1236 (left panel) and L428 (right panel) nuclear extracts. The pull-down by RPA194 antibody (IP), pull-down by empty beads or IgG isotype control, and 1% input protein were loaded for Western blot detection using anti-H2A.B and anti-RPA194 antibodies.

E H2A.B interacts with RPA194 in live cells. Western blot analysis was performed to detect RPA194 in the BioID samples from L1236 cells (left panel), and L428 cells (right panel). The samples were prepared using pull-down with streptavidin beads from the cell lysate of the empty vector, BirA*-H2A.B and H2A.B-BirA* transduced cells, which were preincubated with 50 µM biotin for 24 h. The positive control was loaded as 1% input.

Source data are available online for this figure.