EMBO/soda-nel huggingface dataset:
miRNA profiling of human naive CD4 T cells links miR-34c-5p to cell activation and HIV replication (2016)
Figure 7

Figure 7. miR-34c-5p targets in the HIV interactome.

A. Interaction network between genes regulated by miR-34c-5p and HIV-1 proteins. Figure depicts the subset of DE genes identified in J34c cells that have reported interactions with HIV-1 proteins in the HIV interactome database (Ako-Adjei et al, 2015), as well as their subcellular localization and function (see also Table EV6).

B. Western-blot of PCAF/KAT2B and histone H3 in JØ or J34c (top), confirming decrease in protein levels. Bottom: antagomiR to miR-34c-5p results in increased KAT2B mRNA levels compared to control-treated cells. Plot presents relative expression values for mRNA in 3 biological replicates; bars represent mean and SEM. Differences were found to be significant with p<0.05 (paired two tailed t test).

C. Predicted target sites for miR-34c-5p in the PCAF/KAT2B mRNA(black bars) and miR-target hybridization for the two strongest sites (top); Luciferase assay with the pEZX renilla/firefly-PCAF/KAT2B 3’UTR reporter in HEK293 cells co-transfected with either the miR-34c-5p overexpression vector or pcDNA3 empty control (bottom). Differences were found to be significant with p<0.05 (paired two tailed t test, n=5).
Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import (2020)
Figure 1

Figure 1. AK2 is a substrate of the mitochondrial disulfide relay.

Cysteine conservation of AK2. AK2 contains four cysteines of which three (C40, C42 and C92) are highly conserved. AK2 exists in two isoforms that differ in the C-terminus with the "A" isoform containing C232 and the "B" isoform not. AK2 does not contain a mitochondrial targeting sequence. Logo plot was derived from alignments of AK2 from 163 species.

Interaction analysis for MIA40 and AK2. Stable inducible cell lines that express either different variants of MIA40-Strep (wildtype and a redox inactive mutant (MIA40C53S,C55S), (Fischer et al., 2013) or AK2-HA were treated with N-ethyl maleimide (NEM) to stop thiol-disulfide exchange processes and lysed under denaturing conditions. Lysates were analysed by subsequent affinity purification against Strep (MIA40) and immunoblotting. Like classical MIA40 substrates, AK2 interacted with the active site of MIA40 (lane 6). 0.5% input was loaded as control.

Redox state analysis of endogenous AK2 by inverse redox shift assay. HEK293 cells were lysed by TCA precipitation after NEM treatment, lysates were reduced using TCEP and then thiols modified using the alkylating agent mmPEG12. With this "inverse" method, previously oxidized cysteine thiols are modified with mmPEG12, which results in a slower migration on SDS-PAGE. Treatment of intact cells with diamide and consequently modification of all cysteines served as control. Likewise, absence of NEM treatment served as 'max' control. Treatment lysates with NEM instead of mmPEG12 served as minimal shift (min) control. A, B, isoforms of AK2. Both isoforms of AK2 are semi-oxidized in intact cells.

Oxidation kinetics experiment to investigate maturation of endogenous AK2. HEK293 cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by TCA precipitation, and then the lysate was treated with mmPEG12 to determine the AK2 redox states, followed by IP against AK2. Eluates were analyzed by Tris-Tricine-PAGE and autoradiography. With this "direct" redox state determination method reduced AK2 was modified with either three (isoform B) or four (isoform A) mmPEG12. Semi-oxidized AK2 was modified with only one ("B") or two ("A") mmPEG12, respectively. Unmodified and fully reduced cell lysates served as controls. Endogenous AK2 becomes oxidized with a half time of less than 20 min.

Oxidation kinetics experiment to investigate the influence of MIA40 on AK2 maturation. Experiment was performed as described in (D), except that 72 h before experiments cells were transfected with control siRNA or siRNA directed against MIA40. Endogenous AK2 oxidation depends on MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, ** represents p<0.01 and *** represents p<0.001.
Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import (2020)
Figure 2

Figure 2. Cysteine residues 42 and 94 are critical for mitochondrial accumulation of AK2.

Influence of AK2 cysteines on the AK2 redox state. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were lysed after NEM treatment, and analyzed by inverse redox shift assay using mmPEG12, SDS-PAGE and immunoblot against the HA tag. AK2C42S and AK2C92S are present in their completely reduced state indicating presence of a disulfide bond between both cysteines.

Crystal structure of AK2 (pdb entry 2C9Y). In the crystal structure, a disulfide bond between C42 and C92 is visible with about 50 % occupancy, while 50 % of the Cys42/92 residues are in the reduced state. This disulfide bond is far away from the active site of AK2. The figure was created using PyMOL.

Influence of AK2 cysteines on cellular localization. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were fixed and analyzed by immunofluorescence. The colocalization of AK2-HA variants with Mitotracker was quantified using Cellprofiler (Pearson's correlation coefficient). C42 and C92 are critical for mitochondrial localization of AK2. The C40S mutant exhibits significantly decreased colocalization with mitochondria. A Kruskall-Wallis rank sum test/ Dunn-test was performed. Numbers of analysed cells are indicated in the plot. *** represents p-value <0.001 compared to wild-type. The black horizontal line represents the median. Data information: Scale bars: 10 μm.

Model for AK2 import and oxidative folding. See text for details.
Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import (2020)
Figure 3

Figure 3. AK2 import competes with proteasomal degradation.

Steady state levels of endogenous AK2 in presence and absence of MIA40. 72 h before experiments, HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 or ALR. Cell lysates were analysed by immunoblot against the indicated proteins. MIA40 depletion led to a strong decrease in AK2 and ALR levels but not of the control proteins. ALR depletion did not affect AK2 levels. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.

Stability of endogenous AK2 in the presence and absence of MIA40. 72 h before experiments HeLa, cells were transfected with control siRNA or siRNAs directed against MIA40. Cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by cell lysis, and AK2 was isolated by denaturing immunoprecipitation. Eluates were analyzed by SDS-PAGE and autoradiography. Stability of endogenous AK2 is significantly decreased upon depletion of MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed; * represents p-value <0.05 compared to wild-type.

Stability of cytosolic AK2-HA cysteine variant. Experiment was performed as in (B) except that cells expressing AK2C40S,C42S,C92S-HA were analysed by IP against HA. AK2C40S,C42S,C92S-HA is unstable but becomes stabilized by application of the proteasomal inhibitor MG132. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.

Steady state levels of AK2-HA cysteine variants. AK2C40S,C42S,C92S-HA and AK2WT-HA were expressed in the presence or absence of proteasomal inhibitor MG132. Protein levels at steady state were analysed by immunoblot. All variants are stabilized by proteasomal inhibition. Reported values are the mean of 3 (AK2WT-HA) or 5 (AK2C40S,C42S,C92S-HA) independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. *** represents p<0.001.

Steady state levels of AK2-HA cysteine variant. 72 h before experiments HEK293 cells were transfected with control siRNA or siRNAs directed against the proteasomal subunits PSMB3 and PSMC5. AK2C40S,C42S,C92S-HA was stabilized by decreased proteasome levels. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.
Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import (2020)
Figure 5

Figure 5. Cytosolic AK2 accumulates when DPP9 processing is prevented.

Cell fractionation upon inhibition of DPP9 activity. HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO. Cells were subjected to fractionation into 'cytosol' (supernatant) and 'mitochondria' (pellet) by differential digitonin lysis and centrifugation. The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration (lanes 2,5,8,11) irrespective of 1G244 treatment. Likewise, the cytosolic marker protein LDH localized under all conditions to the same fractions. Conversely, endogenous AK2 appeared at this digitonin concentration in the 'cytosol' fraction but only upon 1G244 treatment (compare lanes 2 and 5). Thus, inhibition of DPP9 results in appearance of AK2 in the cytosol. Quantification shows loading-control adjusted AK2-signals of 1G244-treated 'cytosol' fraction (lane 5) over DMSO-treated 'cytosol' fraction (lane 2). White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed.

Localization of DPP9-insensitive AK2-HA variants. Experiment was performed as described in Figure 2C. Compared to the wild type, DPP9-insensitive and import-competent AK2-HA variants co-localized with cytosolic markers indicating partial cytosolic localization. The colocalization of AK2-HA variants with Mitotracker was quantified using Fiji (Pearson's correlation coefficient). A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. ** represents p<0.01. Data shown in Figures 2C, 5B, S2 and S5 are derived from the same experiments. The black horizontal line represents the median. Data information: Scale bars: 10 μm.

Localization of AK2C40S-HA upon proteasomal inhibition. Experiment was performed as described in Figure 2C. Upon MG132 treatment, colocalization of AK2C40S-HA with the cytosolic marker increased. A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. *** represents p<0.001.The black horizontal line represents the median. Data information: Scale bars: 10 μm.
Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import (2020)
Figure 6

Figure 6. Cytosolic AK2 is active.

AK2 activity assay with varying AMP concentrations. Pyruvate kinase (PK) and lactate dehydrogenase (LDH) thereby couple the oxidation of NADH to the interconversion of adenine nucleotides. The concentration of ATP is maintained constant, whereas the concentration of AMP is titrated.

AK2 activity assay with varying ADP concentrations. Hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) thereby couple the reduction of NADP+ to the interconversion of adenine nucleotides. The concentration of ADP is titrated.

AK2 activity in dependence of its substrate AMP. Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0.012 mM AMP. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are superimposable. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.

AK2 activity in dependence of its substrate ADP. Maximal activity was observed at around 1-2 mM ADP with an apparent KM of 0.12 mM and 0.1 mM ADP for AK2C40S,C232S and AK2C40S,C42S,C92S,C232S, respectively. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are thus highly similar. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.

Activity assay for AK2-HA variants isolated from HEK293 cells. Indicated AK2-HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants. Then, the AK2 activity assay described in (B) was performed. While import-competent AK2 variants exhibited full activity, a cytosolic AK2 variant with the native N-terminus showed very low activity. This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9-insensitive cytosolic AK2 variants. The enzymatic inactive K28Q variants served as negative control indicating that only AK2-HA-dependent activity ways measured by our assay. Reported values are the mean of 2 independent experiments; error bars represent ±SD.

Model for the cytosolic quality control pathway controlling cellular AK2 level and its localization. Details see text.
Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import (2020)
Figure 7

Figure 7. DPP9 controls levels and localization for multiple mitochondrial proteins.

In silico assessment of the mitochondrial proteome ('MitoCarta 2.0', (Calvo et al., 2016)). The MitoCarta was searched for the following DPP9 recognition motifs: MPX (X not P) or MYPX (Y not D, X not P). More than 100 MitoCarta proteins fulfil this criterion. Of those, many do not carry an N-terminal mitochondrial targeting sequence (MTS) but instead internal or C-terminal targeting signals. Proteins found in IMS and IMM appear to be enriched.

In silico assessment of N-termini of human disulfide relay substrates. Approximately 40-50 substrates of the human disulfide relay have been described (Habich et al., 2019b). Of those, 13 contain conserved DPP8/9 processing sites at their N-terminus. For COX17 and NDUFA8, the predicted neo-N-termini 'GL-' (COX17) and 'GI-' (NDUFA8) after DPP9 processing were identified in the mouse N-terminome (Calvo et al., 2017). Logo plots represent alignments over163 different species for AK2 and 11 different species for all other proteins. For many disulfide relay substrates, the cleavage sites for DPP8/9 are conserved.

Steady state levels of NDUFB10, NDUFS5, NDUFA8, COA6, CHCHD2, and TIMM9 upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. Proteins except TIMM9 are stabilized upon DPP9 inhibition. Reported values are the mean of 3-5 independent experiments; error bars represent ±SD. Student's t-test was performed. ** represents p<0.01.

Steady state levels of NDUFB10 variants upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. NDUFB10C107S is stabilized upon DPP9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed for the respective control and treatment samples. ** represents p<0.01.

Localization of NDUFB10 variants upon 1G244 treatment. Experiment was performed as in (C) except that mitochondria were isolated and the fractions analysed by immunoblot against the indicated proteins. Mitochondria contain increased NDUFB10C107S levels after DPP9 inhibition. Reported values are the mean of 2 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.

Expanded View Figures
The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis (2021)
Figure 2

Figure 2. The effect of T cell depletion on tumor onset and progression in 99LN-BrM.

(A) Experimental design of T cell depletion by αCD4 and αCD8a antibody treatment.

(B) Representative flow cytometry blots of blood samples from mice of both groups 3 weeks after treatment start showing successful depletion of CD45+CD3+ T cells in the αCD4+αCD8 treatment group.

(C) Representative IHC images of CD3+ T cells and Iba1+ macrophages/MG in the isotype and αCD4+αCD8 group. Scale bar; 100 μm.

(D) Quantification of Iba1+ and CD3+ cells in IHC sections of BrM from the isotype (n=9) and αCD4+αCD8 group (n=7).

(E) Representative MRI pictures of BrM in early, medium and late stage of both groups.

(F) Kaplan-Meier curves depict BrM free survival of mice in the isotype and αCD4+αCD8 group (Isotype n=19, αCD4+αCD8 n=19).

(G) Tumor growth curves depict the increase of absolute BrM volume for each mouse in both groups over time (Isotype n=19, αCD4+αCD8 n=19).
(H) Heatmap displays the relative expression level of T cell effector and exhaustion marker in TIL-CD4 and TIL-CD8 compared to blood lymphocytes. Values display the average per group based on vst values (BL-CD4 or BL-CD8 with n=3 for each group) vs. BrM-associated TIL-CD4 or TIL-CD8 (n=3 for each group).

Data information: Numerical data in (D) is represented as scatter dot plot with line at mean ± SD. P-values were obtained by unpaired t test in (D) or based on adjusted p value (padj) obtained by Wald test and corrected for multiple testing using the Benjamini and Hochberg method in (H) with *P<0.05, **P<0.01, and ***P<0.001. Exact p-values can be found in Appendix Table S3 for (D) and the Data Source file associated with Figure 2 for (H).
The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis (2021)
Figure 3

Figure 3. Expression of immune checkpoints in breast-to-brain brain metastases.

(A) Surface expression of PD-1 in vivo in 99LN-BrM quantified by flow cytometry (n=3).

(B) PD-L1 in 99LN BrM quantified by flow cytometry (n=3).

(C) Flow cytometric analysis of PD-L1 in tumor-free brain and 99LN-BrM (n=3).

(D) Flow cytometric analysis of PD-L1 expression on 99LN-BrM associated myeloid cell types. (n=6).

(E) Expression of genes associated with antigen presentation and co-regulatory factors in TAM-MG and TAM-MDM. n=3 per condition. Values are depicted as normalized counts. Genes labeled with # can have stimulatory effects depending on the co-regulatory receptor on T cells.

(F) Gene expression of co-regulatory receptors in TIL-CD4 and TIL-CD8. n=3 per condition. Values are depicted as normalized counts.

(G) Functional gene annotation of altered cellular pathways in TAM-MG vs. TAM-MDM. Left panel depicts pathways enriched in TAM-MG, right panel depicts pathways enriched in TAM-MDM. Adjusted p value (padj) were obtained by Wald test and corrected for multiple testing using the Benjamini and Hochberg method.

(H) Representative HALO spatial plot showing the spatial distribution of cell types with indicated marker combination in 99LN-BrM and representative IF images of multiplexed histology. Scale bar; 100 μm.

(I) HALO proximity histogram depicts the distance (μm) between the indicated cell types for individual mice (n=3).

Data information: In (A-D), data is represented as scatter dot plot with lines at mean ± SD. In (E+F), data is represented as scatter dot plot with lines at median. P-values were obtained by unpaired t test with *P<0.05, **P<0.01, and ***P<0.001. Exact p-values can be found in Appendix Table S3
The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis (2021)
Figure 4

Figure 4. Immune cells in BrM after WBRT.
(A) Experimental design of tumor initiation and application of WBRT.
(B) Representative MRI pictures of untreated and irradiated mice at d0 and d14 after treatment start.
(C) Quantification of Iba1+ macrophages in IHC sections of 99LN-BrM at trial endpoint (Ctrl n=27, WBRT n=18).
(D) Representative IF images of 99LN-BrM stained for the macrophage marker Iba1 (red) and the MG marker Tmem119 (white). Higher magnification images present areas in the peri-tumor region and in the tumor core. DAPI was used as nuclear counterstain. Scale bar; 100 µm.
(E) Quantification of different myeloid populations in 99LN-BrM tumors with and without irradiation by flow cytometry at d14 (Ctrl n=5, WBRT n=4).

(F) Representative IF images of 99LN-BrM with and without WBRT stained for DCIR2 (red) and EpCAM (green). DAPI (blue) was used as nuclear counterstain. Scale bar; 100 µm.

(G) Quantification of CD11c+MHCII+ immune cells by flow cytometry in 99LN-BrM with and without WBRT.

(H, I) Relative proportion of cDC1 and cDC2 in 99LN-BrM with and without WBRT.

(J) cDC1:cDC2 ratio in 99LN-BrM based on flow cytometry data (H+I).

(K) Representative IHC images of CD3+, FoxP3+ and CD8+ T cells in 99LN-BrM sections with and without WBRT. Scale bar; 100 µm.

(L) Quantification of CD3+ cells in IHC sections of 99LN-BrM. (Ctrl n=26, WBRT n=18).

(M) FoxP3:CD3 ratio in IHC sections of 99LN-BrM. (Ctrl n=25, WBRT n=18).

(N) CD8:CD3 ratio in IHC sections of 99LN-BrM. (Ctrl n=25, WBRT n=18).

(O-Q) Quantification of CD3+, CD4+ and CD8+ T cells in 99LN-BrM tumors with and without irradiation by flow cytometry.

(R) CD4:CD8 ratio in 99LN-BrM based on data (P+Q).

Data information: In (G-J), n=6 for control and n=7 for WBRT. In (O-R), n=10 for control and for WBRT. In (C+E+G-J+L-N, O-R), data is represented as scattered dot plot with line mean ± SD. P-values were obtained by unpaired t test with *P<0.05. Exact p-values can be found in Appendix Table S3
The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis (2021)
Figure 7

Figure 7. Effects of radio-immunotherapy on T cells and myeloid cells in breast cancer derived BrM.

(A) CD45+ immune cells infiltrating 99LN-BrM (n=5 for isotype, n=4 for the other groups).

(B) Flow cytometric analysis of dendritic cell (CD45+CD11c+CD83+) infiltration (n=4 for isotype, n=3 for all other groups).

(C) Quantitative analysis of IHC CD3+ T cells at trial end point. (n=10 for isotype, n=7 for WBRT, n=10 for PD-1, and n=8 for WBRT+αPD-1 group).

(D) Flow cytometric analysis of T cell subpopulations in 99LN-BrM (n=4 for isotype, n=3 for all other groups).

(E) Quantitative analysis of IHC FoxP3+ T cells at trial end point. (n=10 for isotype, n=7 for WBRT, n=10 for PD-1, and n=8 for WBRT+αPD-1 group).

(F) Flow cytometric analysis of PD-1 expression on T cells in response to different treatments (control n=4, all other groups n=3).

(G) Composition of the myeloid compartment in 99LN-BrM in the different treatment groups (n=5 for isotype, all other groups n=4).

(H) Relative abundance (%) of PD-L1+ myeloid cell types in BrM samples of the four treatment groups (n=5 for isotype, all other groups n=4).

(I) Experimental design of the in vitro T cell activation assay.

(J) Relative CD69 protein level on T cells cultivated with different cell types, unstimulated or stimulated with tumor conditioned media (Cond.) (n=3).

(K) Relative Gzmb and IFNγ protein level on CD4+ and CD8+ T cells cultivated with different cell types, with or without aPD-1 and unstimulated or stimulated with tumor conditioned media (Cond.) (n=4 for conditions including BMDM, n=5 for all other conditions).

Data information: Data in (A-C, E+F) is represented as scattered dot plot with line at mean ± SD. Data in (D+G+H) is represented as stacked columns ± SD. Data in (J+K) is represented as scattered dot plot with line at median. Dotted line depicts baseline T cell activation. P-values were obtained by Mann Whitney test in (C) unpaired t test in (F), two-way ANOVA in (D, G and H) and paired t test in (J and K) with *P<0.05 and **P<0.01, and ***P<0.001. Exact p-values can be found in Appendix Table S3.