EMBO/soda-nel huggingface dataset:
Non-genetic and genetic rewiring underlie adaptation to hypomorphic alleles of an essential gene (2021)
Figure 5

Figure 5. Upregulation of KPNB1 and KPNA4 underlie early adaptation to NUP58 mutations.

Combined median fold-change in RNA (A) and protein (B) levels between NUP58 mutant clones and WT cells (calculated using n=4 RNA-seq and n=3 mass spectrometry datasets). Data were visualized using Cytoscape. White: comparable average expression to WT; red: upregulated relative to WT; blue: downregulated relative to WT. Nodes belonging to the same subcomplex are grouped; edges represent physical interactions (see M&M for details). Transcripts and protein abundance changes in individual clones are reported in Fig EV3.

Diagram detailing experimental method for experiment performed in Fig 5D. A stable cell line carrying KPNB1-KPNA4-eGFP and a WT cell line were transfected with eRFP Cas9 plasmid carrying dual gRNA. Cell populations were either treated with doxycycline (doxy +) or left untreated (doxy -) and individually sorted into 384-well plates. A total of 2 plates were prepared for each replicate.

Cellular viability expressed as number of viable colonies following Cas9-induced gene editing of NUP58 in WT cell line and in a cell line stably overexpressing KPNB1-KPNA4-eGFP (KAPs). The experiment was performed using three independent replicates and as described in Fig 5C. Error bars indicate mean with SD (Welch's t test; **P = 0.0017, *P = 0.0325).
Non-genetic and genetic rewiring underlie adaptation to hypomorphic alleles of an essential gene (2021)
Figure 6

Figure 6. NUP58 mutant isoforms are stabilized upon fitness gain

Maximum growth rates of NUP58 mutant clone C5 during long-term culture. Each dot represents a technical replicate (n=22). Error bars indicate mean with SD (Welch's t test, ns: non-significant, **P = 0.0030, ****P ≤ 0.00001).

Improvement in active nuclear import upon cell passaging. Analysis was performed as in Fig 2C. Dots represent analysed cells (WT P3 n=16, WT P19 n=25, C5 P1 n=24, C5 P3 n=27, C5 P19 n=23) of one independent experimental replicate performed. Error bars indicate mean with SD (Welch's t test; **P = 0.0037, ****P ≤ 0.00001).

Representative images of analysed C5 P1 (left) and C5 P19 (right) NLS-3xmCherry stable cells. Mean intensity of fluorescence for cytoplasm and nucleoplasm for each cell are labelled. Scale bar, 20 μm.

Cytoscape visualization of protein abundance changes between C5 P19 and C5 P1. Red nodes indicate proteins that were upregulated upon passaging; blue nodes indicate proteins that were downregulated upon passaging. White nodes display proteins with comparable expression levels at both early and late passage.

NUP58 protein expression in C5 at P1 and P19. Blots were performed using Antibody 1 (Ab1, top) or Antibody 2 (Ab2, bottom). Actin was used as loading control. The band corresponding to the full-length NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk.

Alternative NUP58 isoform localizes to the NPC. Representative images of HCT116 cell line showing localization of WT NUP58 protein (top WT isoform, z-stack = 10) and of an isoform lacking exon 4 and ending at exon 13 (bottom mutant isoform, z-stack = 11). Scale bar, 20 μm

Cellular viability following Cas9-induced gene editing of native NUP58 (left) or NUP85 (right) in WT cell line (black circles) and in a cell line overexpressing NUP58 mutant isoform (grey circles). The experiment was performed using three independent experimental replicates. Error bars indicate mean with SD (Welch's t test; *P = 0.0194, ns = non-significant).
Non-genetic and genetic rewiring underlie adaptation to hypomorphic alleles of an essential gene (2021)
Figure 7

Figure 7. NUP58 is stabilized upon focal amplification of its coding sequence

Comparison of NUP58 mRNA abundance between late (P19) and early (P1) passage in wild-type cells (WT, top) and clone C5 (bottom). mRNA was quantified using qPCR and five independent probe sets amplifying different regions of NUP58 mRNA. Annealing of the probe sets is indicated at the bottom of the NUP58 structure. Relative mRNA abundance between P19 and P1 is color-coded for each exon analysed (according to the color bar shown below). Red triangles highlight the position of the identified mutations in C5.

Diagram at the top represents G-banding pattern of chromosome 13 and highlights chromosome locations of tested genes (NUP58, GRK1, ADPRHL1). Graph at the bottom reports qPCR results for indicated genes preformed on the gDNA of indicated samples at the indicated passage number. The experiment was performed using three independent experimental replicates. Error bars indicate mean with SD (2-way ANOVA; *P = 0.02, **P = 0.0083, ****P ≤ 0.00001, ns = non-significant).

Representative images of NUP58 gene-specific FISH hybridization for control HAP1 cell lines (WT P3-WT P19) and mutant C5 at P19. Each dot represents a NUP58 locus of cluster thereof. Scale bar, 31.3 μm
Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity (2021)
Figure 1

Figure 1. Platinum and cisplatin activate TLR4 in vitro.

A Activity of an NF-κB reporter relative to vehicle control in human embryonic kidney cells that express TLR4 (hTLR4) or an isogenic control cell line that does not express TLR4 (null2) and were stimulated with vehicle (veh), 1 ng/mL LPS, 400 μM nickel chloride, or 25, 50 or 100 μM platinum(II) chloride and platinum(IV) chloride (n=3 independent biological replicates).

B As per panel (A) but secreted IL-8 was monitored as a metric of TLR4 activation upon stimulation with 50 pg/mL LPS, 200 μM nickel chloride, 100 μM platinum(II) chloride or 100 μM platinum(IV) chloride (n=4 independent biological replicates).

C IL-8 secretion in HEK-hTLR4 and HEK-null2 cells following treatment with cisplatin at the indicated concentrations (n=3 independent biological replicates).

D IL-8 secretion in HEK-hTLR4 cells pre-treated with 4 μM TAK242 (TLR4 inhibitor) or vehicle, and subsequent treatment with 50 pg/mL LPS, 200 μM nickel chloride or 25 μM cisplatin (n=3 independent biological replicates). Mock cells were not subject to pretreatment prior to agonist addition.

Data Information: For all panels actual individual data from each experiment are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers. Statistical analyses were assessed by 2-way ANOVA: in A) hTLR4 compared to null2 cells; in B) agonist treatment compared to non-treated (nil); in C) comparisons between successive concentrations; and in D) comparisons between vehicle and TAK-242 treatments. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test, A, B, D; Tukey test, C).
Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity (2021)
Figure 2

Figure 2. Cisplatin activation of TLR4 is independent of the TLR4 co-receptor, MD-2.

A IL-8 secretion in HEK cells stably expressing hTLR4 but not MD-2 (HEK-isoTLR4), transfected with empty vector (EV) or MD-2 and left untreated (nil) or treated with 1 ng/mL LPS, 200 μM nickel chloride, 2 μg/ml HMGB1, 100 μM platinum(II) chloride, 100 μM platinum(IV) chloride or 25 μM cisplatin (n=3 or 4 independent biological replicates).

B Fold IL-8 secreted (relative to nil treatment) in MD-2 deficient HeLa cells treated with 10 or 100 ng/mL LPS or 25 μM cisplatin (n=4 independent biological replicates).

C IL-8 secretion in HeLa cells transfected with non-targeting (siNT) or TLR4-targeting (siTLR4) siRNA and left untreated (nil), or treated with 30 μM cisplatin (n=3 independent biological replicates). Mock cells were not subject to siRNA treatment prior.

Data Information: Actual individual data are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers (A, C) or mean and standard deviation (B). Statistical analyses were determined in comparison to nil treatments using 2-way (A, C) or one-way (B) ANOVA. ns, not significant; *, P<.05; ****, P<.0001 (Dunnett's test).
Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity (2021)
Figure 3

Figure 3. Deletion of Tlr4 in a murine ear outer hair cell line (HEI-OC1) reduces cisplatin-induced ototoxic responses.

A-D HEI-OC1 cells containing a Tlr4 deletion (Tlr4-/-) were compared to HEI-OC1 non-targeting (NT) control cells and assessed for cell viability (A), AnnexinV/propidium iodide staining (B), ROS generation (C) and IL-6 secretion (D) following cisplatin treatment at the indicated concentrations (n=3 independent biological replicates).

Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to NT at the same cisplatin concentration were assessed by 2-way (A, B) or one-way (C, D) ANOVA. *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001(Bonferroni test).
Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity (2021)
Figure 4

Figure 4. Cisplatin has a primary role in Tlr4 activation in HEI-OC1 cells.

A IL-6 secretion in HEI-OC1 cells treated with 100 pg/mL LPS or 20μM cisplatin (n=3 or 4 independent biological replicates).

B IL-6 secretion in TLR4-/- cells transfected with empty vector (EV) or mouse Tlr4 (mTlr4) following treatment with 20μM cisplatin (n=6 independent biological replicates).

C Il6 and Tlr4 transcript levels in HEI-OC1 cells following treatment with 20μM cisplatin for the indicated times (n=3 independent biological replicates).

Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test).
Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity (2021)
Figure 5

Figure 5. Zebrafish Tlr4 mediates cisplatin-induced otoxicity in vivo.

A Hair cell viability in larval zebrafish (assessed by DASPEI staining) following cisplatin treatment at the indicated concentrations.

B Hair cell viability in larval zebrafish pre-treated with control-, tlr4ba- and/or tlr4bb-targeting morpholino oligonucleotides (MO) and subsequently treated with 15 μM cisplatin.

Data information: In both panels each data point represents a score of hair cell integrity in an individual animal (taken from multiple samples per animal) with lines representing mean and standard deviation. Statistical comparisons between cisplatin concentrations (A) or to control morpholino (B, except as indicated in blue) were assessed by one-way ANOVA. *, P<.05; **, P<.01; ****, P<.0001 (Tukey test).
Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity (2021)
Figure 6

Figure 6. Small molecule inhibition of Tlr4 mitigates cisplatin-induced ototoxic responses in HEI-OC1 cells.

A IL-6 secretion in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 10 ng/mL LPS or 20μM cisplatin (n=5-7 independent biological replicates).

B ROS generation in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 20μM cisplatin (n=3 independent biological replicates).

Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by 2-way (A) or one-way (B) ANOVA. *, P<.05; **, P<.01; ****, P<.0001(Dunnett's test).

Expanded View Figure Legends
Tyrosination of α-Tubulin Controls The Initiation of Processive Dynein-Dynactin Motility (2016)
Figure 1

Figure 1. Role of tubulin tail domains in DDB movement. (A) Representative kymographs showing tetramethylrhodamine (TMR)-labeled DDB movement on each type of recombinant MT lattice, as indicated. Scale bars, 5 μm and 25 sec. Pink arrowhead marks a diffusive DDB complex. See also Movie EV1. (B) Quantification of DDB velocity on MT lattices lacking each of the tubulin-CTTs (n ≥ 100 DDB complexes per condition from at least three independent experiments). No statistical difference was observed between WT and mutant MTs (P ≥ 0.1, unpaired t test) (C-D) Quantification of the number of processive (C), or diffusive (D) DDB complexes relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Processive (unidirectional motion exceeding 0.5 μm) and diffusive (back-and-forth motion) events were scored in a field of view and then divided by the lengths of MTs and time, thus normalizing to a value of number of motility events per μm MT per sec. Error bars SD. (E-F) TIRF images of 1 nM TMR-labeled GST-hDyn (2 mM ATP) (E), or 0.3 nM TMR-labeled p150 (F) molecules (green) bound to either Δα-CTT (top), or Δβ-CTT MTs (bottom). Pink arrows show mutant MTs in both the raw, and SD map images, highlighting differences in binding between WT and mutant MTs that are apparent in the SD map. Right, standard deviation maps from an entire time sequence reveal ensemble binding and dissociation events that lead to variations in pixel intensities (see Methods). Scale bars are 5 μm. (G) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. Image shows first frame from time series. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD. P value (unpaired t test) of Δα-CTT vs. Δβ-CTT MTs = 0.5208 for GST-hDyn, and ≤ 0.0001 for p150.
Tyrosination of α-Tubulin Controls The Initiation of Processive Dynein-Dynactin Motility (2016)
Figure 2

Figure 2. Requirement of α-tubulin tyrosination for processive DDB movement. (A) TIRF image of TMR-labeled DDB molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. A standard deviation map of the whole time sequence is shown, revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs (pink arrow). Representative kymographs of DDB movement are shown from each type of MT present in the same chamber. Scale bars are 5 μm and 25 sec. See also Movie EV2. (B-C) Quantification of the number of processive (B) or diffusive (C) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber (n ≥ 20 MTs quantified for each condition from three independent experiments). Error bars represent SD. Data from Δα-CTT MTs (Fig. 1) are re-plotted for comparison. (D) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs. Data points from two independent experiments are shown. (E) TIRF images of 0.3 nM TMR-labeled p150 (top) or 1 nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown. Scale bars are 5 μm and 25 sec. (F) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2 mMATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT. (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.
Tyrosination of α-Tubulin Controls The Initiation of Processive Dynein-Dynactin Motility (2016)
Figure 3

Figure 3. Role of tyrosination in DDB movement on brain MTs. (A) Immunoblot of brain tubulin with or without carboxypeptidase A (CPA). Note CPA specifically removes the α-tubulin C-terminal tyrosine without affecting other PTMs. (B) TIRF image of TMR-labeled DDB molecules (green) on either WT (red) or CPA-treated (blue) pig brain MTs. Standard deviation map of the entire time sequence shows DDB preference for WT MTs and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec. (C) Quantification of the number of processive DDB complexes per μm MT per sec on the CPA-treated MTs, relative to WT MTs in the same chamber (n ≥ 7 MTs of each type quantified from at least three independent experiments). Error bars represent SD. (D) TIRF images of 0.3nM TMR-labeled p150 (top) or 1nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either WT (red), or CPA-treated MTs (blue). Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec for p150, and 5 μm and 12 sec for GST-hDyn (E) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2mM ATP) or p150 bound to CPA-treated MTs relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.
Tyrosination of α-Tubulin Controls The Initiation of Processive Dynein-Dynactin Motility (2016)
Figure 4

Figure 4. Role of the p150 CAP-Gly domain in recognition of α-tubulin tyrosination. (A) Schematic of the p150 constructs used. The p135 isoform lacks the CAP-Gly domain, but retains part of the adjacent basic domain (red) and coiled-coil domains (blue). Right, Coomassie blue stained gel of the purified p150 and p135 proteins. Molecular weight markers are indicated. (B) TIRF image of either p150 (0.3 nM), or p135 (0.3 or 6 nM) bound to either WT (red), or CPA-treated porcine MTs (blue). Note: no binding of p135 is observed at comparable concentrations to p150 indicating the lower binding affinity of this isoform. Proteins were visualized by fluorescence from the GFP tag fused to each construct. (C) Still images from a TIRF time-series of p135 (green) binding to either WT (red), or CPA-treated porcine MTs (blue). Right, standard deviation map of p135 binding shows no preference for either type of MT, and quantification of mean fluorescence intensity (arbitrary units) per μm MT for p135 bound to CPA-treated MTs relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from three independent experiments). Scale bars are 5 μm.
Tyrosination of α-Tubulin Controls The Initiation of Processive Dynein-Dynactin Motility (2016)
Figure 5

Figure 5. α-Tubulin tyrosination is required for the initiation, but not continuation of processive DDB movement. (A) Schematic showing the assembly of a chimeric MT lattice created by end-to-end annealing of stabilized MTs. The MTs are differentially fluorescently labeled prior to annealing. (B) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated (red) and detyrosinated (blue) sections. Most TMR-labeled DDB molecules (green) move processively through the annealed junction between the two types of MT lattice (65%, n=836 processive complexes, 65 MT-MT junctions, three independent experiments). Diffusive DDB molecules (white arrow) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary. Right, video frames showing a processive DDB complex (pink arrowhead) moving across the junction (yellow arrowhead) of tyrosinated and detryosinated MT. The (+) and (-) signs denote MT polarity inferred from the directionality of DDB movement. Scale bars are 5 μm and 25 sec. See also Movie EV3. (C) TIRF images of low (0.3 nM) and high (10 nM) concentrations of TMR-labeled p150 (green) bound to chimeric yeast MTs containing tyrosinated (red) and detyrosinated (blue) sections of MT. Representative kymographs below show diffusive behavior of p150 molecules on the MT. Note that p150 rarely diffuses into the detyrosinated section of MT, even at high concentrations. Scale bars are 5 μm and 10 sec. (D-E) TIRF images and kymographs of annealed WT and CPA-treated porcine MTs (D), or annealed WT MTs (E). The majority of DDB complexes (78%, n=960 processive complexes, 45 MT-MT junctions, three independent experiments) move processively across the boundary between WT and CPA-MTs (D). Note TMR-labeled DDB complexes (green) in (E) traverse the annealed boundary without pausing (93%, n=677 processive complexes, 45 MT-MT junctions, three independent experiments), suggesting that the annealed junction does not affect DDB motility. (F) TIRF image and kymograph from a chimeric MT composed of WT (red) and subtilisin-treated porcine MTs (blue). Note some DDB complexes (pink arrows) traverse the boundary and continue moving along the subtilisin-treated section of the MT (41%, n=709 processive complexes, 56 MT-MT junctions, 3 independent experiments). All scale bars in D-F are 5 μm and 25 sec.
Long-lived macrophage reprogramming drives spike protein-mediated inflammasome activation in COVID-19 (2021)
Figure 1

Figure 1: SARS-CoV-2 Spike protein induces IL-1β secretion in macrophages from COVID-19 patients.

A SDS PAGE of the recombinant SARS-CoV-2 spike protein (S-protein).

B SARS-CoV-2 spike binding assay of IgGs isolated from COVID-19 patients (n = 3; red circles) or SARS-CoV-2 naïve individuals (SC-naïve) (n = 2; blue circles).

C Experimental scheme: After PBMCs isolation, CD14+ cells were enriched by positive selection. Subsequently, 5x104 CD14+ cells/well were seeded and incubated in the presence of M-CSF for 5 days. Differentiated macrophages were stimulated with/without recombinant SARS-CoV-2 spike protein or lipopolysaccharide (LPS) for 4h. To activate IL-1β secretion nigericin or ATP was added for 2h. Finally, IL-1β secretion was quantified by ELISA.

D Quantification of IL-1β concentration (pg/ml) in the supernatant of primary macrophage cultures from COVID-19 patients (n = 44; red bars) or SC-naïve individuals (n = 24; blue bars) stimulated with LPS or S-protein (0.1 µg/ml). To activate IL-1β secretion nigericin was added for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used.

E TNF-α (pg/ml) secreted from macrophages derived from SC-naïve and COVID-19 patients as described above. Cells were stimulated with LPS (SC-naïve/COVID-19 n = 4), LPS with nigericin (SC-naïve n = 4; COVID-19 n = 6), S-protein (SC-naïve n = 5; COVID-19 n = 4), S-protein with nigericin (SC-naïve n = 3; COVID-19 n = 4) or left unstimulated (SC-naïve n = 3; COVID-19 n = 4). Significances shown in the figure are always in comparison to the unstimulated control.

For statistical analysis, two-way ANOVA with tukey post hoc test was used.

Data information: Graphs show mean ± SEM. **p<0.01; ****p<0.0001.