EMBO/soda-nel huggingface dataset:
Cyclin D3 restricts SARS-CoV-2 Envelope incorporation into virions and interferes with viral spread. (2022)
Figure 2

Figure 2

Proteasome inhibition abolishes effect of SARS-CoV-2 infection on D-cyclins depletion

A549 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 18h post-infection. Cells were lysed 24h post-addition of inhibitor and cyclins and viral proteins were detected by western blot. (N) nucleocapsid.

Densitometry analysis of western blots for D-cyclins (normalized to actin) in A549 AT2 cells. Plots are average of 2 (cyclin D1), 3 (cyclin D3) biological replicates. Bars indicate mean with SD. Statistical analysis was performed using ordinary two way ANOVA; ns, non-significant; ∗p < 0.1.

A459 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 8h post-infection. Cells were fixed and stained 24h post-addition of inhibitor. (N) nucleocapsid.Scale bars: 40μm. Contour lines represent outlines of infected cells.

Quantification of D3 cyclin relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells have been counted. Bars indicate mean with SD. Statistical analysis was performed using ordinary two-way ANOVA; ∗∗∗p < 0.001.
Cyclin D3 restricts SARS-CoV-2 Envelope incorporation into virions and interferes with viral spread. (2022)
Figure 3

Figure 3

Cyclin D3 impairs SARS-CoV-2 spreading infection

(A-D) (A,B) D and A-cyclins were depleted using siRNA in A549 AT2 cells. Cells were infected 18h later with Delta (∆), Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.001, 0.1, 0.1 respectively. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for western blots (A) and TCID50 (B). (C,D) D and A-cyclins were depleted using siRNA in VERO AT2 cells. Cells were infected 18h later with Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.1. Cells were washed 4h post-infection and new media added. Supernatants and cells were collected at 24h and 48h later. (A,C) Representative example of western blot from uninfected cell lysates collected at 72h post-knockdown. (B,D) Virus titres in cell culture supernatants were determined as TCID50 in VERO AT2 cells.

Cyclin D3 was depleted using siRNA in Calu3 cells. Cells were infected 48h later with

Delta SARS-CoV-2 at MOI 0.01. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for western blot and TCID50. Uninfected cell lysates were used for western blot.

Data information: Graphs represent average of n=3 (B,D); n=4 (E) biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.
Cyclin D3 restricts SARS-CoV-2 Envelope incorporation into virions and interferes with viral spread. (2022)
Figure 5

Figure 5

SARS-CoV-2 mediated depletion of D-cyclins is cell cycle arrest independent

(A-D) A549 AT2 cells were depleted for D and A2 cyclins and 18h later infected with Delta variant SARS-CoV-2 for 24h. Cells were fixed, stained for SARS-CoV-2 nucleocapsid and analysed for infection and Fucci cell cycle sensor. (A) A representative western blot from lysates of uninfected knock-down cells. (B) Example of gating strategy for flow cytometry analysis. (C) Percentage of infected cells in cells depleted for cyclins. n=3 biological replicates; Ordinary two-way ANOVA with Sidak's multiple comparisons test: ns, non-significant; ∗∗∗∗p < 0.0001; ∗∗p < 0.01; ∗p < 0.1. Bars indicate mean with SD. (D) Flow cytometry analysis of early S and S/G2/M cell cycle phases comparing cyclin D1, D3, and A2 knockdown to NT (non-target siRNA). n = 3-4 biological replicates. Statistical analysis was performed using two-sided unpaired Student's t-tests; ns, non-significant; ∗∗p < 0.01; ∗p < 0.1. Bars indicate mean with SD.

(E) A549 AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and VSV-G pseudotyped lentiviral particles containing WT cyclin D3 or T283A mutant cyclin D3 (mutant not degraded by proteasome). Cells were infected 24h later and collected 48h post-infection for flow cytometry analysis of early S and western blot. n=2 biological replicates; Ordinary two-way ANOVA with Sidak's multiple comparisons test: ns, non-significant; ∗∗p < 0.01. Bars indicate mean with SD.

(F,G) VERO AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and 18h later infected with Delta variant SARS-CoV-2. Cells were fixed and stained for D-cyclins 24h later. (F) Example of acquisition using automated microscopic platform. Cells are identified for infection, expression of cyclin D3, and cell cycle (Red/arrow=G1phase; Green/arrowhead=S/G2/M; Red+Green/arrowhead=early S). Scale bar: 20μm. (G) Quantification of D-cyclins re-localization from nucleus to cytoplasm and correlation with cell cycle phases using ImageJ and Harmony (PerkinElmer). At least 50-200 cells were analysed in each condition. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ∗p < 0.1.
Cyclin D3 restricts SARS-CoV-2 Envelope incorporation into virions and interferes with viral spread. (2022)
Figure 6

Figure 6

Cyclin D3 associates with SARS-CoV-2 protein E and M

(A) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E or nsp9, and control plasmid (EV). Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA, and cyclin D3 antibodies.

(B) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E, M, both E and M or nsp9. Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA antibodies. WCL, whole cell lysate.

(C-G) 293T GFP11 cells were transfected with Spike, and/or with Envelope, Membrane, and cyclin D3. 24h post-transfection cells were seeded at a 1:1 ratio with Vero-GFP10 cells and percentage of GFP+ve area (syncytia) were determined 18h later. S, Spike; E, Envelope; M, Membrane; D3, cyclin D3.

(C,D) Representative images of GFP+ syncytia. Scale bars: 40μm.

(E-G) Quantification of cell-to-cell fusion showing percentage of the GFP+ve area to the acquired total cell area. (E) n=4 biological replicates. (F,G) n = 4 biological replicates; technical triplicates shown; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01 . Bars indicate mean with SD.

(H) Diagram of SARS-CoV-2 VLPs construction in the presence or absence of cyclin D3 in 293T cells.

(I) Representative example of western blot from purified SARS-CoV-2 Virus Like Particles (VLPs) and whole cell lysates (WCL). MCM2 was used as loading control.

(J) Quantification of protein incorporation (into VLPs) or expression (in WCL). n = 4 biological replicates; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗p < 0.001. Bars indicate mean with SD. N, nucleocapsid; M, membrane; E, envelope; S, spike.

Expanded View Figures
Human TLR8 senses UR/URR-motifs in RNA derived from bacteria and mitochondria (2015)
Figure 4

Figure 4 – Lysosomal function inhibition affecting TLR8 activity is anti-inflammatory upon both, Gram-positive and Gram-negative bacterial infection ex vivo.

A Cytokine release of 3 days differentiated THP-1 cells of indicated genotypes upon TLR2 blockade (T2.5) and challenge with TLR ligands, heat inactivated (hi) or viable bacteria.

B PBMC activity upon endosome function inhibition (chloroquine) and TLR2 blockade (T2.5) followed by TLR ligand challenge or bacterial infection.

C Activity of whole blood culture upon endosome function inhibition (chloroquine) and challenge with TLR ligands, heat inactivated (hi), or viable bacteria.

A-C Graphs show mean ± SD; n = 3 each; *, p ≤ 0.05; **, p ≤ 0.01; -, unchallenged; P3C, Pam3CSK4; n.d., not detected; triangles, decreasing doses of exponentially growing bacteria gentamicin treated 1 h post infection.
Neighboring mutation-mediated enhancement of dengue virus infectivity and spread (2022)
Figure 2

Figure 2. The impact of stable substitutions in the DENV E protein on viral infectivity.

A-D One-week-old mosquitoes were thoracically infected with DENV2-Asian I (A), DENV2-Asian American (B), DENV3-I (C), and DENV4-I (D) mutants, respectively. A. aegypti mosquitoes were infected intrathoracically with 10 p.f.u. of DENV. The DENV viral loads were assessed at 3 days postinfection via qRT-PCR and were normalized against A. aegypti actin.

E-H Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV2-Asian I (E), DENV2-Asian American (F), DENV3-I (G), and DENV4-I (H) were measured by qRT-PCR and were normalized against mouse GAPDH.

I, J Infection of DENV2-Asian I mutants on hu-moDC (I) and hu-moMØ (J). The cells were infected with 0.01 MOI of the indicated viruses. The infected cells were detected at 48 h postinfection by qRT-PCR, and DENV viral loads were normalized against human GAPDH.

Data information: In (A-D), one dot represents one mosquito, and the horizontal lines represent the median of the results. In (E, G-J), n = 6 biological replicates. In (F), n = 5 biological replicates. In (E-J), data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test (A-H) and unpaired t-test (I, J). The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).
Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models (2021)
Figure 2

Figure 2. Neither erlotinib nor 2XmAbs are effective, but the combination cures a xenograft model driven by a single-mutation EGFR.

PC9 cells (3X106/mouse) were subcutaneously implanted in the flanks of CD1-nu/nu mice. When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, 0.2 mg/mouse/injection), once every three days, or with erlotinib (50 mg/kg/day), once per day. Alternatively, mice were treated with combinations of erlotinib (50, 20, 10, 5 or 1 mg/kg) and the two monoclonal antibodies. Following 60 days of treatment, we reduced the frequency of erlotinib administration to once every other day (underneath dotted line).

A, B Tumor volumes (A) and animal survival (B) are shown. Mice were euthanized when tumor size reached 1,500 mm3. Data are means ± SEM from 5-9 mice per group.

(C) Statistical analysis of tumor volumes corresponding to the last measurement for each mouse was performed using one-way ANOVA followed by Tukey's multiple comparison test. Results are shown as mean ±SEM. See p-values and number of mice per group (N) in Appendix Table S2 Note that only the significant comparisons are shown.

(D) Shown are tumor volumes of individual mice of each group. Note that each animal is represented by a different color. The respective numbers of tumor-free mice are indicated. The grey areas represent treatment phases and they are followed by drug holidays (blank).
Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models (2021)
Figure 3

Figure 3. A combination of mAbs specific to EGFR and to HER2 collaborates with both second- and third-generation EGFR TKIs, but a BCR-ABL TKI displays no cooperative effects.

PC9 cells (exon 19 deletion) were subcutaneously implanted in the flank of CD1-nu/nu mice (3X106/mouse). When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, each at 0.1 mg/mouse/injection) once every three days, or daily with different TKIs: osimertinib (5 mg/kg), afatinib (5 mg/kg) or imatinib (100 mg/kg), either alone or in combination with the two antibodies. Following 60 days of treatment, the frequency of TKI administration was reduced to once every other day (underneath dotted lines), while mAb treatment remained unaltered.

A, B Tumor growth (A) and animal survival (B) are shown. Mice were euthanized when tumor size reached 1,500 mm3. Data are means ± SEM from 5-9 mice of each group.

(C) Statistical analysis of tumor volumes corresponding to the last measurement for each mouse was performed using one-way ANOVA followed by Tukey's multiple comparison test. Results are shown as mean ±SEM. See p-values and number of mice per group (N) in Appendix Table S2. Note that only the significant comparisons are shown.

(D) Shown are tumor volumes corresponding to individual animals of each group. The numbers of mice with undetectable tumors are indicated. Note that the control and 2XmAbs arms are shared with Figure 2. The grey areas represent treatment phases and they are followed by drug holidays (blank).
Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models (2021)
Figure 4

Figure 4. Animal treatments with combinations of 2XmAbs plus EGFR-specific TKIs induce downregulation of survivin and several receptors for growth/survival factors.

CD1-nu/nu mice carrying PC9 xenografts were divided in groups (2-3 mice/group) and treated for seven days with different TKIs: erlotinib (50 mg/kg or 20 mg/kg), osimertinib (5 mg/kg) or afatinib (5 mg/kg), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection). Note that when singly applied, we used erlotinib at 50 mg/kg.

(A) Shown are fold changes in tumor volume at day 7 of treatment. The horizontal line indicates no change in tumor volume (fold change) between days 0 and 7 of treatment.

(B) After treatment, all mice were sacrificed and tumors extracted. Protein extracts were resolved by means of electrophoresis and transfer to nitrocellulose membranes, which were later incubated overnight with the indicated antibodies. This was followed by incubation with peroxidase-conjugated secondary antibodies. Signals were detected using ChemidocTM (from Bio-Rad), quantified and normalized to the signals of GAPDH or tubulin (numbers shown below each lane).
Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models (2021)
Figure 5

Figure 5. Combination treatments comprising a mixture of two antibodies (cetuximab and trastuzumab) and either erlotinib or osimertinib overcome resistance to TKI in an animal model.

A, B PC9 cells (3X106/mouse) were subcutaneously injected in the flanks of CD1-nu/nu mice. When tumors reached a volume of approximately 500 mm3, mice were treated daily with erlotinib (50 mg/kg, blue area; panel A) or with osimertinib (5 mg/kg, grey area; panel B) using oral gavage. Initially, all tumors displayed stable disease or they regressed, but eventually all started relapsing. Once relapsing tumors reached 800 mm3, while under erlotinib or osimertinib treatment, we supplemented the treatment with a combination of 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection; twice a week) plus either erlotinib (50 mg/kg; A, red area) or osimertinib (5 mg/kg; B, orange area). Tumor volumes of individual mice are shown.
Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models (2021)
Figure 6

Figure 6. PDX models corresponding to the most frequent founder EGFR mutations can be effectively inhibited by combinations of antibodies and TKIs; relapsing tumors remain sensitive to erlotinib+2XmAbs even after a long drug holiday (>3 months).

A, B NSG mice were pre-implanted with tumor fragments derived from two different PDX models: (A) TM00199 (L858R-EGFR; from The Jackson Laboratory) or (B) TM00193 (E746_A750 del-EGFR; from The Jackson Laboratory). Once tumors reached approximately 300 mm3, mice were treated with monoclonal antibodies (2XmAbs, cetuximab plus trastuzumab, 200μg/injection) twice a week, or with EGFR TKIs, erlotinib (50mg/kg) or osimertinib (10mg/kg) daily. Alternatively, mice were treated with a combination of 2XmAbs and either erlotinib or osimertinib, for either 32 days (A) or 42 days (B). Tumor growth was monitored twice a week. Notice that the antibodies were injected into the peritoneum and the TKIs were delivered orally. The figure shows tumor volumes of individual mice and the respective survival curves (right hand panels). The grey areas mark time windows of animal treatment.

(C) Mice presented in the erlotinib+2XmAbs group in B (left panel, bottom row) were re-treated with the same drug combination (2XmAbs plus erlotinib) after a holiday period (>136 days), during which they received no treatment. Note that different colors identify individual mice from the corresponding panel of B.
Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models (2021)
Figure 7

Figure 7. By blocking FOXM1 and pathways involved in mitosis and cytokinesis, a combination of two antibodies and erlotinib overcomes TKI resistance in an animal model.

(A) A scheme depicting the experimental plan and longitudinal sampling.

(B) For RNAseq analyses, PC9 cells (3X106/mouse) were subcutaneously injected in the flanks of 13 CD1-nu/nu mice. When tumors reached 500 mm3, mice were treated daily with erlotinib (50 mg/kg). Once tumors that initially responded to erlotinib monotherapy started relapsing and reached 800 mm3, we switched to a combination of 2XmAbs plus erlotinib (50 mg/kg). The respective tumor volumes are presented in Figure EV5-A. Control mice (N=5) were sacrificed when tumor volumes reached approximately 500 mm3 (orange star in A). Mice responding to erlotinib (N=3) were sacrificed after one week of treatment (green star in A). Mice resistant to erlotinib (N=3) were sacrificed when tumor volumes reached 800 mm3 (blue star in A), whereas mice responding to erlotinib+2XmAbs (N=2) were sacrificed after one week of treatment (purple star in A). RNA was extracted from all 13 tumors and utilized for RNA-seq analysis. Differentially expressed (DE) genes in the Erlotinib (Er)+2XmAbs arm compared to the Control group are presented in the heatmap.

(C) Protein extracts were prepared from all tumors and analyzed using immunoblotting, as indicated. Signals were quantified and normalized to the signals of GAPDH (numbers shown below each lane).

(D) Summary model: ERK and AKT phosphorylate the transcription factor FOXO3a, thereby dampen its ability to downregulate FOXM1. The combination of erlotinib, cetuximab and trastuzumab overcomes TKI resistance by inhibiting ERK, AKT and FOXM1 transcription. This downregulates several FOXM1 target genes, such as cyclinB1, AURKA, CENPA, BIRC5, PRC1 and KIF4A. All these genes play an essential role in the late steps of the cell cycle, primarily mitosis and cytokinesis, and their downregulation results in inhibition of cell proliferation. The lower panel refers to the tumor samples analyzed in B and shows the calculated fold changes of a gene module repressed after treatment with erlotinib+2XmAbs.

Expanded View Figure legends
Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat (2010)
Figure 9

Autophagy in sav mutants. (A) EM scan of a 7‐day‐old sav3 mutant ommatidium and zoom‐in on autophagosomes containing undigested debris (arrowhead). Scale bar: 1 μm for panel on the left and 0.2 μm for zoom‐in panel on the right. (B) Graph of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 1‐week‐old sav3 clones. Significant accumulation of AV is found in mutant cells. ***P0.001 in one‐tailed t‐test. N=45 pigmented cells versus 42 non‐pigmented cells. (C) Model for the relationship between Atro, Ft/Hippo and neuronal homeostasis. Atro wt overexpression, and more effectively polyQ Atro overexpression, causes a downregulation of ft transcription and of other genes important for photoreceptor neuron function and homeostasis. This results in downregulation of Ft/Hippo signalling, which affects neurodegeneration. Healthy Ft/Hippo signalling contributes to neuronal homeostasis through effective autophagy. Atro may also exert Ft‐independent effects through other factors (like Tetraspanins) and polyQ Atro also affects neurons through polyQ‐specific factors (like misfolding and aggregation), which put additional pressure on the autophagic system. Other polyQ proteins may not feed into the Ft/Hippo pathway, as in the case of Htt‐ex1‐93Q in flies.
THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer (2021)
Figure 1

Figure 1: Loss of THEM6 impairs PCa tumour growth following ADT.

A, Volcano plot of the differentially modulated proteins in 22rv1 (CRPC) versus CWR22res (HN) tumours. Red and blue dots represent the proteins that are significantly up- and down-regulated, respectively (p-value < 0.05, FC = 1.5).

B, Western blot analysis of THEM6 expression in CRPC (22rv1 and LNCaP AI) and HN (CWR22res and LNCaP) prostate orthografts. VCL was used as a sample loading control.

C, IHC staining of THEM6 expression in CWR22res and 22rv1 orthografts. Scale bar represents 100 µm.

D, Western blot analysis of THEM6 expression in CTL and THEM6 KO CRPC cells. HSC70 was used as a sample loading control.

E, Proliferation of CTL and THEM6 KO CRPC cells after 72 hours. Data are expressed as a percentage of CTL cells.

F, Cell stiffness (Young's modulus) of CTL and THEM6 KO CRPC cells measured by atomic force microscopy.

G, Tumour volume (measured by ultrasound) of CTL and THEM6 KO 22rv1-derived orthografts developed in surgically castrated mice.

H, Tumour volume of CTL and THEM6 KO LNCaP AI-derived xenografts developed in surgically castrated mice.

I, Tumour volume (measured by ultrasound) of CTL and THEM6 KO CWR22res-derived orthografts. Orchidectomy was performed 3-weeks after cell implantation.

Data information: Panels E, G, H, I: Data are presented as mean values +/- SD. Panel F: Centre line corresponds to median of data, top and bottom lines correspond to maximal and minimal values. Statistical analysis: E, F: 1-way ANOVA with a Dunnett's multiple comparisons test. G, H: two-tailed Mann-Whitney U test. I: Kruskal-Wallis test. Data reproducibility: Panel A: n = 3 tumours per group. Panel B: n = 1 gel loaded with three prostate orthografts per condition. Panels C, D: representative image from 3 independent biological experiments. Panel E: n = 3 independent biological experiments. Panel F (up): n = 46 (CTL); 47 (KO1); 49 (KO2) cells measured. Panel F (down): n = 31 (CTL); 43 (KO1); 50 (KO2) cells measured. Panels G, I: n = 5 mice per group. Panel H: n = 7 mice per group.