EMBO/soda-nel huggingface dataset:
Distinct retrograde microtubule motor sets drive early and late endosome transport (2020)
Figure 6

Figure 6. Concerted antagonistic action of dynein and KIFC1 controls early endosome retrograde transport.

(A) Confocal microscopy images of untreated (UT) or treated with Ciliobrevin D (CilioD) or AZ82 or both treatments simultaneously (CilioD + AZ82) ECs and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and Draq5 (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.

(B) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3 and EV5) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during AZ82 treatment in ECs, compared to untreated cells (UT). Results are from two independent experiments for a total of 853 early endosomes in 6 untreated cells (142 ± 22 endosomes per cell) and 355 early endosomes in 7 AZ82 cells (51±12 endosomes per cell). Data are analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.

(C) Distribution of retrograde velocity overtime, quantified by image (data correspond to movies EV3, EV4 and EV5) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during Ciliobrevin D (CilioD D) or AZ82 treatment in ECs, compared to untreated cells (UT). Results are from three independent experiments for a total of 580 EEs in 6 UT cells (97 ± 18 endosomes per cell), 432 EEs in 9 CilioD cells (48 ± 15 endosomes per cell) and from two independent experiments for a total of 223 EEs in 7 AZ82 cells (32 ± 6 endosomes per cell). Data are analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and CilioD treated ECs p≤ 0,01 ** and for UT and AZ82 treated ECs p≤ 0,001 ***.

(D) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3, EV5 and EV6) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during AZ82 treatment (as in b), alone or in combination with CilioD treatment (CilioD + AZ82), compared to untreated EC cells (UT). Results are from two independent experiments for a total of 853 early endosomes in 6 UT cells (142 ±22 endosomes per cell), 355 EEs in 7 AZ82 cells (51 ± 12 endosomes per cell) and 1106 EEs in 8 CilioD+AZ82 cells (136 ± 42 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and AZ82 treated ECs p≤ 0,001 *** (orange) and for CilioD and CilioD + AZ82 treated ECs p≤ 0,001 *** (green).
Distinct retrograde microtubule motor sets drive early and late endosome transport (2020)
Figure 8

Figure 8. STIM1 controls late endosome pH, mTORC1 signaling and autophagy.

(A) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), to which the pH-sensitive dye pHrodo Green was given and then cells were stained for LAMP-1 (in red) to visualize LEs and DAPI (in blue) to highlight the nucleus. Scale bar = 20 μm.

(B) Relative amount of LAMP-1+ LEs positive to the pH-sensitive dye pHrodo (as in a) in shCTL and shSTIM1 ECs. Results are the average ± SEM of three independent experiments for a total of 90 cells (30 cell for experiment) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.

(C) Representative Western Blot analysis of endogenous Raptor, total and phospho-Thr389 S6K1 proteins (normalized on total tubulin levels) in ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (siSTIM1). Numbers above each band represent respective N.O.D. average (two biological replicates) quantification.

(D) Representative Western Blot analysis of endogenous LC3-I and -II proteins (both normalized on total tubulin levels and represented as ratio LC3-II/LC3-I) in ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (siSTIM1). Results are the average ± SD of three independent experiments and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,01 **.
Viral Bcl-2-mediated evasion of autophagy aids chronic infection of gammaherpesvirus 68. (2009)
Figure 2

vBcl-2 Interaction with Beclin1.

(A) Co-immunoprecipitation (Co-IP) of Beclin1 with WT or mutant vBcl-2. 293T cells were transiently transfected with the indicated constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of V5-tagged Beclin1. (B) Co-IP of WT or mutant vBcl-2 with endogenous Beclin1. 293T cells were transfected with the indicated vBcl-2 constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of endogenous Beclin1. 1% whole-cell lysates (WCLs) was used as the input. (C) vBcl-2 interaction with Bak protein. 293T cells were transfected with WT and mutant forms of vBcl-2 as indicated. At 48 h posttransfection, WCLs were mixed either with GST-BakΔTM fusion protein (left panel) or with GST alone (right panel) for an in vitro GST pull-down (GST PD) assays. GST fusion proteins used for the pulldown assay are indicated (bottom panel). 1% WCL was used as the input. Data are representative of at least three experiments yielding similar results.
Viral Bcl-2-mediated evasion of autophagy aids chronic infection of gammaherpesvirus 68. (2009)
Figure 3

Anti-autophagic Activity of the vBcl-2 Mutant Proteins.

(A) NIH3T3 cells stably expressing the WT and mutant forms of vBcl-2 were transfected with GFP-LC3. At 18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as mean±SEM of the combined results from three independent experiments. **, P<0.0001. (B) NIH3T3 cells expressing the WT or mutant forms of vBcl-2 as indicated were transfected with GFP-LC3 and treated with 2 µM rapamycin for 6 h. GFP-LC3 was detected using an inverted fluorescence microscope (top). Autophagy was quantified as mean±SEM of the combined results from three independent experiments. Scale bar, 5 µm; **, P<0.01. (C) NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2, as indicated, were treated with 2 µM rapamycin. LC3-I and LC3-II levels were then determined by immunoblotting with an antibody against LC3 (top). Densitometric quantification of the LC3-II/LC3-I ratios under normal and rapamycin treatment conditions is shown at the bottom. Similar results were obtained from three independent experiments.
Viral Bcl-2-mediated evasion of autophagy aids chronic infection of gammaherpesvirus 68. (2009)
Figure 4

The Beclin1-binding α1 Helix of vBcl-2 is Required for Inhibition of Autophagy during γHV68 Infection.

(top) Representative confocal images of GFP-LC3 and HA-tagged vBcl-2 (WT and mutants) in NIH3T3 cells infected with the indicated viruses (MOI = 5). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Scale bar, 5 µm. (bottom) Quantification of the percentage of virally infected cells with GFP-LC3 punctate staining. Results shown represent mean±SEM of combined results from three independent experiments (200 cells per experimental condition). *, P<0.01 versus HA-WT; **, P<0.001 versus HA-WT.
Viral Bcl-2-mediated evasion of autophagy aids chronic infection of gammaherpesvirus 68. (2009)
Figure 5

Anti-apoptotic Activities of the vBcl-2 Mutant Proteins.

NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B) or for the caspase 3 activation using flow cytometry (C). Apoptotic cells in (B) were counted under high power magnification (60×magnification). Mock, untreated condition. Data represents mean±SEM of combined results from three independent experiments. Scale bars, 100 µm (A), 5 µm (B). **, P<0.0001 versus vector cells.
Viral Bcl-2-mediated evasion of autophagy aids chronic infection of gammaherpesvirus 68. (2009)
Figure 6

In Vitro and In Vivo Lytic Replication of the WT and Recombinant γHV68 Viruses.

(A) Single-step (right) and multiple-step (left) growth curves of the WT and recombinant γHV68 viruses in BHK21 cells. (B) Acute replication of the WT and mutant vBcl-2 γHV68 viruses in the lungs of BALB/c mice at 5 dpi (up) and 7 dpi (down) after intranasal infection determined by viral titers in the lungs of the infected mice (left) or by real-time PCR of the viral genomic DNA (right). Mean±SEM of five mice per group/experiment. Data of 7 dpi is pooled from two separate experiments. The vBcl-2 Δα1 and ΔBH2 mutants did not yield significantly different results when compared to the WT in infectious virus titers [for Δα1, P = 0.82 (day 5); P = 0.08 (day 7); for ΔBH2, P = 0.49 (day 5); P = 0.54 (day 7); unpaired t-tests] and in viral genome loads in the lungs [for Δα1, P = 0.25 (day 5); P = 0.28 (day 7); for ΔBH2, P = 0.21 (day 5); P = 0.37 (day 7); unpaired t-tests]. n.s., not significant.
Viral Bcl-2-mediated evasion of autophagy aids chronic infection of gammaherpesvirus 68. (2009)
Figure 7

Distinct Roles for the vBcl-2-mediated Inhibition of Autophagy and Apoptosis in γHV68 Chronic Infections In Vivo.

Splenic infectious centers measured at 12 dpi (A, left), 14 dpi (B, left), 21 dpi (C, left), 28 dpi (D, up), 35 dpi (E, up), or 42 dpi (F, up) and viral genome load measured by real-time PCR at 12 dpi (A, right), 14 dpi (B, right), 21 dpi (C, right), 28 dpi (D, down), 35 dpi (E, down), or 42 dpi (F, down), in the BALB/c mice intranasally infected with the WT or recombinant γHV68 mutants, as indicated (Mean±SEM of five mice per group/time point/experiment). Data of 14 dpi and 28 dpi is pooled from two and three separate experiments, respectively. Preformed infectious virus was negligible in all spleen samples. No significant difference was detected at 12 dpi (A), 14 dpi (B), and 21 dpi (C) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [for Δα1, P = 0.58 (day 12); P = 0.75 (day 14); P = 0.18 (day 21); for ΔBH2, P = 0.64 (day 12); P = 0.73 (day 14); P = 0.81 (day 21); unpaired t-tests] and in viral genome loads [for Δα1, P = 0.85 (day 12); P = 0.76 (day 14); P = 0.96 (day 21); for ΔBH2, P = 0.56 (day 12); P = 0.73 (day 14); P = 0.25 (day 21); unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased infectious center titers (top) of vBcl-2 mutant viruses, as compared to WT γHV68, were statistically significant [vBcl-2Δα1 versus vBcl-2AAA (42 dpi), P = 0.46; vBcl-2Δα1 versus WT γHV68 (42 dpi), P = 0.08; unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased viral DNA loads (bottom) of the vBcl-2Δα1, vBcl-2AAA, and vBcl-2 null mutants, as compared to WT γHV68 and the vBcl-2ΔBH2 mutant, were statistically significant as follows (unpaired t-tests): at day 28, vBcl-2Δα1 versus WT γHV68, P<0.01; vBcl-2AAA versus WT γHV68, P<0.01; vBcl-2-null versus WT γHV68, P<0.01; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2-null versus vBcl-2ΔBH2, P<0.001; at day 35, vBcl-2Δα1 versus WT γHV68, P<0.05; vBcl-2AAA versus WT γHV68, P<0.05; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.01; vBcl-2AAA versus vBcl-2ΔBH2, P<0.05; at day 42, vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2ΔBH2 versus WT γHV68, P = 0.33. I.C., infectious center. *, P<0.05; **, P<0.01; ***, P<0.001.
Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development (2020)
Figure 1

Fig. 1. The mitotic surveillance pathway is conserved in the mouse in vivo.

(A) Gross morphology of control (Ctrl), Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text. Scale bar = 500 μm.

(B, C) Immunostaining for p53 (B) and Cleaved-Caspase3 (Cl-CASP3, C) on transverse sections of Ctrl, Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. The area shown encompasses the ventral neural tube and surrounding mesenchyme. Asterisks in (B) denote non-specific staining of blood cells. Scale bar = 50 μm.

(D, E) Quantifications of the p53 (D) and Cl-CASP3 (E) fluorescence signals shown in (B) and (C), respectively. Three embryos per genotype were used for the quantifications. *** p < 0.001, *p<0.05 (two-tailed student's T-test, one-way ANOVA with Tukey's multiple comparisons tests gave similar results). Bars represent mean ± s.d. (D) Ctrl: 1.00 ± 0.06 (n=1770 cells); Sas-4−/−: 6.84 ± 0.34 (n=275), Sas-4−/− 53bp1−/−: 1.85 ± 0.75 (n=690), Sas-4−/− Usp28−/−: 1.50 ± 0.48 (n=975). (E) Ctrl: 1.00 ± 0.70 (n=1690); Sas-4−/−: 7.88 ± 2.62 (n=175); Sas-4−/− 53bp1−/−: 1.43 ± 0.71 (n=1030); Sas-4−/− Usp28−/−: 1.57 ± 0.44 (n=950).
Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development (2020)
Figure 2

Fig. 2. p53 upregulation in Sas-4−/− embryos is evident by E7.5 and is not due to the onset of 53BP1 or USP28 expression.

(A, B) Whole-mount immunostaining and quantification for p53 on Ctrl and Sas-4−/− embryos at E7.5 (A) and E6.5 (B). Representative sagittal planes are shown and the dotted lines demarcate the epiblast. Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below, normalized to control embryos in the same batch at E7.5 (4 embryos) and at E6.5 (9 embryos). *** p < 0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bars = 100 μm. (A) Ctrl: 1.04 ± 0.00 (n=495 cells); Sas-4-/-: 1.65 ± 0.19 (n=850). (B) Ctrl: 1.00 ± 0.18 (n=1665); Sas-4-/-: 1.16 ± 0.44 (n=1330)

(C, D) Immunostaining for 53BP1 and USP28 on sagittal sections of WT embryos at E6.5 (C) or sagittal planes of whole-mount immunostaining at E5.5 (D). Quantification of the fluorescent signals in the epiblast (Ep) versus visceral endoderm (VE) are shown below. Four embryos per genotype were used for the quantifications. **** p < 0.0001, ***p<0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 50 μm. (C) 53BP1 in Ep: 1.00 ± 0.10 (n=225 cells); 53BP1 in VE: 0.37 ± 0.06 (n=105); USP28 in Ep: 1.00 ± 0.08 (n=235); USP28 in VE: 0.17 ± 0.05 (n=90). (D) 53BP1 in Ep: 1.00 ± 0.24 (n=225); 53BP1 in VE: 0.68 ± 0.14 (n=115); USP28 in Ep: 1.00 ± 0.23 (n=280); USP28 in VE: 0.28 ± 0.00 (n=110).
Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development (2020)
Figure 3

Fig. 3. Sas-4−/− mESCs activate the mitotic surveillance pathway.

(A) Immunostaining for the centrosome marker γ-tubulin (TUBG) on Ctrl and Sas-4−/− primary mESCs. The bottom panels are magnifications of the areas marked in the top panels. Scale bars = 20 μm (top) and 10 μm (bottom).

(B) Immunostaining and quantification for p53 on Ctrl and Sas-4−/− primary mESCs in pluripotent and partially differentiated (diff.) conditions. The quantification of p53 fluorescence intensities was normalized to Ctrl (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test, the comparison of the genotypes in the "Pluripotent" condition was not significant using the one-way ANOVA with Tukey's multiple comparisons tests). Bars represent mean ± s.d. Scale bar = 50 μm. Pluripotent Ctrl: 1.00 ± 0.02 (n=2030 cells); pluripotent Sas-4-/-: 1.17 ± 0.10 (n=2170); partially diff. Ctrl: 0.62 ± 0.13 (n=2745); partially diff. Sas-4-/-: 1.12 ± 0.24 (n=2000).

(C, D) Three-day growth curves of WT and Sas-4−/− (C) or p53−/− and Sas-4−/− p53−/− (D) primary mESCs in the indicated conditions starting with 105 cells on Day 0 (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test). Bars represent mean ± s.d. For details of the measurements, see Materials and Methods (Tables 3 and 4).
Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development (2020)
Figure 4

Fig. 4. Centrioles gradually mature during mouse development.

(A) Sagittal planes of whole-mount immunostaining and quantification for TUBG on WT mouse embryos from E3.5 to E6.5. The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass (E3.5) or epiblast (E5.5 and E6.5). Bars represent mean ± s.d (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests). Scale bars: E3.5 and E5.5 = 15 μm; E6.5 = 30 μm and 3 μm (insets). E3.5: 88 ± 26% (n=73 cells from 5 embryos); E5.5: 92 ± 10% (n=270 from 3 embryos); E6.5: 79 ± 12% (n=12,082 from 3 embryos).

(B, C) Representative gSTED super-resolution images of immunostaining for TUBG and either CEP164 (B) or ODF2 (C). Whole-mount staining was used for the CEP164 data as well as for the E3.5 blastocysts for ODF2, whereas staining on sections was used for ODF2 at E5.5 and E6.5. Quantification of the percentage of centrosomes (TUBG) with CEP164 or ODF2 are shown on the right. **** p < 0.0001, *** p < 0.001, * p < 0.05 (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests; the ODF2 data between E5.5 and E6.5 was not significant using the latter test). Bars represent mean ± s.d. Scale bar = 0.5 μm. CEP164 at E3.5: 2 ± 2%, (n=92 centrosomes from 4 embryos); E5.5: 50 ± 2% (n=249 from 3 embryos); E6.5: 62 ± 0% (n=856 from 3 embryos). ODF2 at E3.5: 20 ± 9% (n=108 from 5 embryos); E5.5: 51 ± 4% (n=294 from 5 embryos); E6.5: 60 ± 2% (n=1,183 from 4 embryos).

(D) Immunostaining and quantification of the area of TUBG and PCNT signals from E5.5 and E6.5 centrosomes in epiblast cells. The separate channels for the marked centriole are shown in the insets. *** p < 0.001, ** p < 0.01 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 2 μm. Quantification of PCNT at E5.5: 0.22 ± 0.01 μm (n=103 centrosomes from 3 embryos); E6.5: 0.31 ± 0.02 μm (n=342 from 3 embryos). Quantification of TUBG at E5.5: 0.15 ± 0.02 μm (n=108 from 4 embryos); E6.5: 0.25 ± 0.01 μm (n=311 from 3 embryos).
SARS-CoV-2 infection remodels the host protein thermal stability
landscape (2021)
Figure 1

Figure 1. Global assessment of host abundance and thermostability changes during SARS-CoV-2 infection.

a) Caco-2 monolayers consisting of 1.5e6 cells per sample were infected with SARS-CoV-2 at an MOI of 0.5 (see materials and methods). Three biologically independent replicates were collected per time point. Samples were subjected to vacuum manifold-based 2D-TPP. After aggregate removal, the remaining soluble fractions were labelled with isobaric mass tags (TMTpro) to enable protein quantification. Samples were combined in a 2D-TPP layout in order to compare protein stability and abundance throughout the infection time course (Becher et al, 2018). The uninfected (t-1) time point was used as a reference to calculate fold changes (FC). Significant protein changes (abundance and thermal stability) are represented as circle plots for individual proteins, as previously described (Becher et al, 2018). Inner circle corresponds to protein abundance and the outer circle corresponds to stability changes (n=3).

b) Caco-2 cells were infected with SARS-CoV-2 as per Figure 1A. Cells were washed, fixed and stained with DAPI (blue) to visualise cell nuclei and incubated with anti-N protein (red) to visualise SARS-CoV-2 infected cells. Scale bar denotes 10 µM.

c) Infection rates were calculated using automated imaging software (see materials and methods) calculated from six fields of view per time point per biological replicate. Data point indicates the sample means and the error bars the standard error of the mean (SEM) (n=3).

d) SARS-CoV-2 detected proteins plotted as a function of time where each data point denotes a different replicate (n=3).
SARS-CoV-2 infection remodels the host protein thermal stability
landscape (2021)
Figure 4

Figure 4. Thermal stability changes in SARS-CoV-2-targeted host proteins.

a) IMPDH2 was consistently destabilized from 4 hours post SARS-CoV-2 infection. IMPDH2 was previously shown to form a protein-protein interaction with nsp14 (green line) (Gordon et al, 2020b) and be a crucial factor in supporting SARS-CoV-2 replication (Gordon et al, 2020a). The SARS-CoV-1 nsp14 has a dual role in base pair mismatch repair and synthesis of the Guanine-rich 5' cap of viral RNA (Smith & Denison, 2013; Smith et al, 2015; Chen et al, 2009). Circular stability and abundance plots are from Caco-2 cells infected with SARS-CoV-2 as described in 1A (see also 1A for plot legend). Asterisk denotes significant regulation (see methods).

b) Thermal destabilisation of UGDH and PAAF1 temporally coincide and both were previously shown to interact with the SARS-CoV-2 proteins nsp6 (UGDH), and M (UGDH and PAAF1) (Samavarchi-Tehrani et al, 2020). PAAF1 negatively regulates the proteasome by controlling its assembly/disassembly (Park et al, 2005) and has been previously linked to transcription of HIV-1 (Lassot et al, 2007; Nakamura et al, 2012). Significant protein changes (abundance and thermal stability) are represented as circle plots for individual proteins, as previously described (Becher et al, 2018). Inner circle corresponds to protein abundance and the outer circle corresponds to stability changes (n=3). Asterisk denotes significant regulation (see methods).

c) SAFB phosphopeptide abundance changes during SARS-CoV-2 infection. Vero cell phosphopeptide data from(Bouhaddou et al, 2020). Data previously acquired from Vero cells infected with SARS-CoV-2 (Bouhaddou et al, 2020). S604 phosphosite that temporally coincides with SAFB thermal destabilisation (Potel et al, 2020). Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS-CoV-2 infected Caco-2 cells as described in 1A (n=3). Asterisk denotes significant regulation (see methods).

D) Thermal stability profile for all unmodified SAFB peptides (green) and the phosphorylated S604 (pS604) (purple) in HeLa cells. Data from (Potel et al, 2020).
SARS-CoV-2 infection remodels the host protein thermal stability
landscape (2021)
Figure 5

Figure 5. 2D-TPP abundance and stability changes uncover functionally relevant host targets during SARS-CoV-2 infection.

a) Heat shock proteins, HSP90AA1 and HSP90AB1, were destabilised during SARS-CoV-2 infection of Caco-2 cells (circular plots, left side (n=3); see 1A for circular plot legend). Significant protein changes (abundance and thermal stability) are represented as circle plots for individual proteins, as previously described (Becher et al, 2018). Inner circle corresponds to protein abundance and the outer circle corresponds to stability changes (n=3). Asterisk denotes significant regulation (see methods).

b) The corresponding HSP90 inhibitor, Tanespimycin, effectively suppressed SARS-CoV-2 proliferation (n=2). Calu-3 cells were pre-incubated with the compound, followed by the addition of SARS-CoV-2 and subsequent co-incubation in the presence of the compound for 20-24 hours. Viral load was quantified in parallel using antibody-based detection of double stranded RNA (orange). Compound toxicity in Calu-3 cells was assessed (purple) treated with the compound alone for 20-24 hours.

c) The aryl hydrocarbon hydroxylase (Cyp1a1) increased in abundance between 4-12 hours post infection and was then reduced between 24-48 hours post infection (circular plot, left, (n=3); see 1A for circular plot legend). Circular plots displayed as described in panel a. Asterisk denotes significant regulation (see methods).

d) The Cyp1a1 inhibitor suppressed SARS-CoV-2 proliferation (n=2). Dose response curves were performed as described in 4A. See Appendix Figure S7 for additional tested drugs. Data displayed as described in 5B.