EMBO/soda-nel huggingface dataset:
LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling (2020)
Figure 6

Figure 6. Nutrient starvation reduced the level of LRP6 and its O-GlcNAcylation in vivo.

A. Nutrient starvation reduced the level of LRP6 in mouse liver. Mice were starved for 36 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. **P < 0.01. Student's t-test was used for statistical analysis.

B. Refeeding of nutrients rescued LRP6 in mouse liver. Mice were starved and then refed for 6 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis.

C. The level of LRP6 was not reduced during nutrient starvation in mouse brain. Mice were starved and brain tissues were analyzed by immunoblotting.

D. LRP6 was O-GlcNAcylated in mouse liver. Liver tissues were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.

E. O-GlcNAcylation of LRP6 was reduced during starvation in mouse liver. LRP6 in liver tissue lysates from both fed and starved mice was equilibrated by loading twice as much lysate from the livers of starved mice on the gel. Liver tissues were incubated in sWGA-beads for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.

EXPANDED VIEW FIGURE LEGENDS
Alterations of redox and iron metabolism accompany development of HIV latency (2020)
Figure 2

Figure 2. Activation of Nrf2 drives antioxidant responses to HIV-1 induced oxidative stress.

(A) Total to oxidized glutathione ratio (GSH/GSSG) in mock infected or HIV-1 infected CD4+ T-cells (7 dpi, n=4 biological replicates; mean±SEM).

(B-D) representative picture (B) and quantification (C,D) of ROS content in HIV-1 infected or mock infected primary CD4+ T-cells at 7dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence, HIV-1 DNA+ cells were identified by FISH (D). The black dash in (C) indicates the median. Box plots in (D) depict median and 25-75 percentile, while whiskers extend from min to max. n= number of cells. Scale bar = 2μm.

(E) Subcellular localization of Nrf2 in CD4+ T-cells infected with HIV-1 or mock infected (3dpi) as analyzed by biochemical fractionation and western blot; Whole Cell Extract (WCE), Cytoplasm (Cyt), Nucleoplasm (Nuc).

(F) Nuclear localization (left) and content (right) of Nrf2 in HIV-1 RNA+ and HIV-1 RNA- cells (7dpi) as measured by combining immunofluorescence and HIV-1 RNA FISH. Black dash indicates median, n= number of cells. Scale bar = 2μm

(G, H). Time course of the mRNA (G) and protein (H) levels of main downstream antioxidant targets of Nrf2 during the transition from productive (3-9 dpi) to latent (14 dpi) infection, as measured by qPCR (G) and western blot (H). Data in (G) are depicted as mean±SEM of 3 biological replicates. GCLC and HMOX-1 in (H) were probed upon membrane stripping.

(I) Relative mRNA expression of Nrf2 downstream antioxidant targets in J-Lat 9.2 cells left untreated (latent) or treated for 24h with 10µM TPA (reactivated). Data are depicted as mean±SEM of 3 biological replicates.

Data information: Values shown in (C,D and F) were calculated as nuclear corrected total cell fluorescence (as in(McCloy et al, 2014)) and analyzed by Kruskal-Wallis multiple comparison test followed by Dunn´s post-test (C) or by two-tailed Wilcoxon signed-rank test (D,F). For (G,I), raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in infected vs mock infected cells (G), or TPA-reactivated vs latent cells (I) which were calculated as in (Livak & Schmittgen, 2001). Data were analyzed by two-way ANOVA followed by Tukey´s post-test for multiple comparisons. * P<0.5 ** P<0.01; ***P<0.001; ****P<0.0001. Trx= thioredoxin; NQO1= NAD(P)H [quinone] dehydrogenase 1; HMOX-1= heme oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC= glutamate-cysteine

ligase; TrxR1= thioredoxin reductase 1.
Alterations of redox and iron metabolism accompany development of HIV latency (2020)
Figure 3

Figure 3. HIV-1 replication alters iron metabolism and enhances iron import during transition to latency.

(A) gag mRNA expression (left) and relative viability (right) of HIV-1 infected Jurkat-TAg cells transfected with non targeting siRNA (NC) or siRNAs targeting the HMOX-1 gene. Cells were infected 24hr post-transfection and assayed for mRNA expression (by qPCR) or viability (by MTT assay) 48hr post-infection. Data (mean±SEM; n=3 technical replicates) were normalized using the NC control and analyzed by unpaired t-test. P<0.05; **P<0.01.

(B,C) RNA-Seq analysis of different time points of primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. The gene set used for the analyses is GO iron ion import (12 genes, GO:0097286). Data in (B) were analyzed by Fisher test. Boxplots depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. Dots below boxplots illustrate, for each time point, the pathway enrichment analysis of genes up-regulated in infected vs matched mock-infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of DEGs in the pathway; grey dots are not statistically significant. (C) Heatmaps of the standardized expression of the GO iron ion import pathway genes in HIV-1 infected and mock infected samples over time. Expression levels were standardized [(mean gene expression - SD)/SD] for each gene in each time point. The TFRC (TfR1) gene, which was considered for further analysis, is highlighted.

(D) TfR1 expression in HIV-1 infected or mock infected CD4+ T-cells as visualized by confocal microscopy. Fluorescent values shown were calculated as described in (McCloy et al, 2014). Data were analyzed by one-way ANOVA followed by Sidak´s post-test to compare corresponding mock infected vs infected pairs. Black dash indicates median, n= number of cells. Scale bar = 2μm.

(E) Western blot (upper panel) and relative expression (lower panel) of FTH-1 protein in HIV-1 infected vs mock infected CD4+ T-cells over time. Western blot quantification was performed with Fiji-Image J (Schindelin et al, 2012), normalized to the housekeeping protein beta-actin and expressed as Log2 fold change in HIV-1 infected vs mock infected cells (mean±SEM; n=3 biological replicates). P<0.05;

(F) Percentage of intracellular p24+ gag CD4+ T-cells infected with HIV-1 and left untreated or treated for 48hr with the iron donor Fe-NTA at 150μM concentration (n=3; mean ± range of three biological replicates).

(G) Percentage of GFP+ J-Lat cells (clones 9.2 and 15.4) left untreated or treated for 48hr with TPA (10μM), the iron donor FeNT (150μM) or a combination of the two. Data are expressed as mean±SEM of four technical replicates for each cell line and were analyzed by one-way ANOVA followed by Tukey´s post-test. *P<0.05; ***P<0.001; ****P<0.0001.
Alterations of redox and iron metabolism accompany development of HIV latency (2020)
Figure 4

Figure 4. HIV-1 replication drives depletion of the redox-sensitive marker PML.

(A,B) RNA-Seq (A) or proteomic (B) analyses of different time points in primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. Data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots below boxplots illustrate the pathway enrichment analysis of genes or proteins up-regulated in infected vs matched mock infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes or proteins in the pathway; grey dots are not statistically significant. The gene set considered for the analyses is Biocarta_PML_pathway (17 genes, M4891).

(C,D) Western blot (C) and relative expression (D) of PML protein and mRNA in HIV-1 infected vs mock infected CD4+ T-cells over time. Raw data were normalized using actin (protein) or GAPDH or 18S (mRNA) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells. Data were analyzed by two-way ANOVA followed by Tukey´s post-test (mean±SEM of 3 and 4 biological replicates for PML mRNA and PML protein, respectively).

(E) Western blot and quantification of PML protein in two different clones of J-Lat cells left untreated or treated with TPA for 24h. Data were analyzed by unpaired t-test (mean±SEM; n=3 technical replicates for each cell line).

(F,G) PML protein expression in CD4+ T-cells infected with HIV-1 or mock infected at 7dpi as measured by western blot. Cells were left untreated or treated with the antioxidant compound NAC (10mM, F) or the iron chelator deferiprone (L1, 50μM, G).

Data information: *P<0.05 **P<0.01.
Alterations of redox and iron metabolism accompany development of HIV latency (2020)
Figure 5

Figure 5. PML is preferentially depleted in cells with detectable HIV-1 mRNA.

(A,B) Representative image and quantification of the number of PML NBs in HIV-1 DNA+ vs mock infected (A) and HIV-1 RNA+ vs HIV-1 RNA- (B) CD4+ T-cells. Scale bars = 2μm.

(C) Representative 3D reconstruction and quantification of the number of PML NBs in HIV-1 RNA+ vs HIV-1 RNA- CD4+ T-cells. n= number of cells.

Data information: Infected cells were identified with either DNA (A) or RNA (B,C) FISH and PML NBs were stained by IF. The algorithm used for automatic PML counting and 3D reconstruction is detailed in the Materials and Methods section. Data are depicted as mean±SEM and were analyzed by unpaired t-test. **P<0.01; ***P<0.001; ****P<0.0001.
Alterations of redox and iron metabolism accompany development of HIV latency (2020)
Figure 6

Figure 6. PML degradation is mediated by the ubiquitin-proteasome axis.

(A-D) Representative immunofluorescence images (A,C) and quantifications (B,D) of PML NBs (green, maximal projection) and mab414/nuclear pore (red, one Z stack) in J-Lat 9.2 cells. Cells were left latent or reactivated for 24 hrs with 10μM TPA. Proteasome function was blocked for 4 hrs in both latent and TPA reactivated cells using MG132 (10μM) or Bortezomib (100nM) (A,B). Alternatively, ubiquitination was blocked for 4 hrs using TAK243 (10μM) (C,D). Scale bar 10μm.

(E,F) Representative immunofluorescence images (E) and quantification (F) of PML NBs (green) and SUMO 2/3 (red) in J-Lat 9.2 cells left latent or reactivated for 24 hours with 10μM TPA. Cells were imaged by STED microscopy, scale bar 2 μm.

(G,H) Representative immunofluorescence images (G) and quantification (H) of PML NBs (green, maximal projection) and SUMO 2/3 (red, maximal projection) during mock and productive HIV-1 infection of primary CD4+ T-cells at 7dpi. Scale bar 10μm.

I) Schematic representation of the interplay between metabolic imbalances induced by HIV-1 replication and PML stability. Upon HIV-1 infection of activated CD4+ T-cells, ROS content is increased by HIV-1 replication and potentially by iron through the Fenton reaction, leading to oxidative stress. Oxidative stress is accompanied by PML NBs SUMOylation and PML protein degradation via the ubiquitin-proteasome axis. Upregulation of Nrf2-mediated antioxidant responses in infected cells facilitates cell survival and reversal to a resting state where ROS content is decreased and PML reformed, while latency reactivation reignites oxidative stress and PML degradation.

Data information: Data were analyzed by Kruskal-Wallis test (B,D) or unpaired Mann-Whitney test (F,H). Black dash indicates median, n= number of PML NBs. * P<0.05; ** P<0.01; ****P<0.0001.

Expanded View figures
The human phosphatase CDC14A modulates primary cilium length by regulating centrosomal actin nucleation (2018)
Figure 2

Figure 2. hCDC14A localizes to actin cytoskeleton and the basal body during ciliogenesis

TetON-hCDC14A-GFP RPE1 cells were serum starved for 48 h prior to fixation and immunofluorescence microscopy. hCDC14A-GFP colocalizes with Phalloidin 555 conjugated dye that marks F-actin fibers. The boxed area was 10-fold enlarged and displayed below the corresponding images to indicate co-localization of hCDC14A-GFP and F-actin.

Line scan inside the magenta colored box showed in (A).

Sub-centrosomal localization of hCDC14AC278S-GFP was determined by correlative positioning of the GFP signal with the proteins Arl13B and IFT88 (cilia), C-Nap1/ninein (proximal end), CEP164 (distal appendage) and ODF2/ ninein (subdistal appendage). Results are fitted in the cartoons adjacent to the respective panels for better visualization.

The cartoon summarizes the localization of hCDC14A on cilia. The distal appendage protein CEP164 marks the mother basal body. The C-Nap1 organized centrosome linker connects the mother with the daughter basal body (black lines). ODF2 and ninein mark the subdistal appendages. Note, ninein has a second localization at the distal end of basal bodies. hCDC14A localized to the subdistal appendages and the C-Nap1 marked proximal end of the basal bodies.

TetON-hCDC14A-GFP RPE1 cells were serum starved for 48 h prior to fixation and immunofluorescence microscopy. hCDC14A-GFP colocalizes with Phalloidin 555 conjugated dye that marks F-actin fibers. The boxed area was 10-fold enlarged and displayed below the corresponding images to indicate co-localization of hCDC14A-GFP and F-actin.

Line scan inside the magenta colored box showed in (A).

Sub-centrosomal localization of hCDC14AC278S-GFP was determined by correlative positioning of the GFP signal with the proteins Arl13B and IFT88 (cilia), C-Nap1/ninein (proximal end), CEP164 (distal appendage) and ODF2/ ninein (subdistal appendage). Results are fitted in the cartoons adjacent to the respective panels for better visualization.

The cartoon summarizes the localization of hCDC14A on cilia. The distal appendage protein CEP164 marks the mother basal body. The C-Nap1 organized centrosome linker connects the mother with the daughter basal body (black lines). ODF2 and ninein mark the subdistal appendages. Note, ninein has a second localization at the distal end of basal bodies. hCDC14A localized to the subdistal appendages and the C-Nap1 marked proximal end of the basal bodies.

Data information: (A, C) Sizes of the scale bars are indicated next to the designated images.
The human phosphatase CDC14A modulates primary cilium length by regulating centrosomal actin nucleation (2018)
Figure 5

Figure 5. Phospho-regulation of DBN1 is critical for cilia length control

Schematic representation of different domains of DBN1 (ADF-H: ADF homology domain, AB1: Actin-binding region 1, AB2: Actin-binding region 2, P: Proline-rich region) [44]. The numbers represent the amino acid positions of the denoted domains. Three independent gRNAs targeting different exons (3, 5, 7) of DBN1 were used to generate DBN1 KO cells.

Successful targeting of DBN1 was confirmed by sequencing the PCR amplicons surrounding the locus. For example, in clone 3.3, there was a single base insertion in both DBN1 alleles (T in the one, G in the other) to cause disruption of protein expression through the introduction of a premature stop codon.

The absence of DBN1 protein in the targeted clones was further verified by immunoblotting of the whole cell lysate. GAPDH is the loading control.

One targeted clone from each gRNA (1.16, 2.18, 3.3) including the non-targeted clone 2.19 and WT control were serum starved for 48 h to induce ciliogenesis. Subsequently, cilia length was measured. The cilia from DBN1 KO cells were significantly longer than those from WT or non-targeted controls. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. **** P≤ 0.0001.

Overexpression of DBN1, DBN1S142A and DBN1S142D in RPE1 WT cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. ** P≤ 0.01; *** P≤ 0.001; ns: not specific.

The phospho-inhibitory (S142A) version of DBN1 rescued the elongated cilia of DBN1 KO cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. * P≤ 0.05; *** P≤ 0.001; ns: not significant. Pairwise comparison was done with unpaired student's t-test and the p-value was written in the figure. Global comparison was done with one-way ANOVA and the p-values were marked with stars.

The overexpressed WT DBN1 was hyper-phosphorylated in cells as detected by phospho-specific DBN1pS142 antibodies. The DBN1pS142 antibodies did not detect the overexpressed DBN1S142A or DBN1S142D.

Immunoblot analysis confirming the successful siRNA depletion of CDK5 and indicating hypo-phosphorylation of DBN1S142 in response to CDK5 depletion.

The DBN1pS142/GAPDH ratio from (H) was densitometrically measured. Three independent experiments. Mean ± SD. * P≤ 0.05.

Depletion of CDK5 decreased the cilia length in WT cells. It restored cilia length in hCDC14APD cells. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. **; P≤ 0.01; **** P≤ 0.0001; ns: not significant.

CDK5 mediated cilia length decrease can be completely blocked by expression of the phospho-mimetic DBN1S142D in hCDC14APD cells. Three independent experiments; 150 cells per experiment and condition. Mean ± SD; ns = not significant; **** P≤ 0.0001.

Data information: (D, E, J, K) One-way ANOVA. (I) Unpaired Student's t-test.
The human phosphatase CDC14A modulates primary cilium length by regulating centrosomal actin nucleation (2018)
Figure 6

Figure 6. hCDC14APD cells show enhanced endosome recycling as well as elevated docking of myosin Va vesicles and Arp2 recruitment to the basal body

Transferrin uptake was measured with serum starvation or 4 h after serum starvation as described [17]. Fluorescent transferrin was added for 45 min to the cells. Cells were analyzed after washing. The red circle around the mother centriole marker CEP164 indicates the fluorescence intensity that was determined for (B). The encircled area in MERGE is shown 2-fold enlarged on the right hand side of the figure.

Quantification of (A). The fluorescence intensities of transferrin within a 2 μm-radius circle surrounding the CEP164 signal were quantified. An ImageJ macro was used to measure the intensity as described in methods. Data were calculated from a total of 120 cells pooled from two independent experiments. Mean ± SD. **** P≤ 0.0001.

hCDC14APD cells showed an increased localization of myosin Va containing vesicles within the 1 μm-radius of the basal body (red cicle). The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure.

Quantification of C. Data was calculated from a total of 180 cells pooled from three independent experiments. Mean ± SD. * P≤ 0.05, *** P≤ 0.001, **** P≤ 0.0001.

Arp2 localization in REP1 WT, hCDC14APD and DBN1 KO cells. Signal intensity was measured 30 min after serum starvation. The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure.

Quantification of E. Sub-centrosomal (1 μm-radius; red circle in E) Arp2 was measured by quantifying the intensity of Arp2 in WT, hCDC14APD and DBN1 KO cells. Both hCDC14APD and DBN1 KO cells contain significantly higher Arp2 in the pericentrosomal area (1 μm-radius) compared to WT cells after 30 min of serum starvation. Data were calculated from a total of 120 cells pooled from three independent experiments. Mean ± SD. **** P≤ 0.0001.

Pericentrosomal ARP2 enrichment was comparable in the RPE1 DBN1 KO cells (Tet Control) with DBN1 KO cells expressing different DBN1 versions (WT, S142A, S142D). Data were calculated from a total of 200 cells each pooled from two independent experiments. Mean ± SD. ns: not specific.

Proposed model for primary cilia length regulation by hCDC14A, CDK5 and DBN1 in RPE1 cells.

Data information: (A, C, E) Sizes of the scale bars are indicated next to the designated images. (B, D, F, G) One-way ANOVA.
Aneuploid senescent cells activate NF-κB to promote their immune clearance by NK cells (2021)
Figure 3

Figure 3. Mechanisms triggering senescence contribute to NK cell recognition in ArCK cells

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.

p53 and p21 levels were determined by western blot analysis. Vinculin was used as loading control. Results were comparable between 2 biological replicates.

The degree of senescence was measured by senescence-associated β-galactosidase (β-gal) activity. The graph shows the percentage of β-gal positive cells. At least 100 cells were analyzed per condition per replicate. n=3 biological replicates; mean ± SEM. Ctrl vs. Doxo, ***p= 0.0006; Ctrl vs. Palbo, *p= 0.025; Ctrl vs. Nutlin, *p= 0.016; Ctrl vs. Torin, p= 0.806, n.s.; Ctrl vs. ArCK, ***p= 0.0001; unpaired t-test.

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.