EMBO/soda-nel huggingface dataset:
Androgen receptor-regulated circFNTA activates KRAS signaling to promote bladder cancer invasion (2020)
Figure 4

Figure 4. circFNTA regulates invasion and cisplatin chemoresistance by competing with miR-370-3p to modulate its host gene FNTA.

(A) Western blot analysis showed that shcircFNTA can decrease FNTA expression in UMUC3 cells (upper panel) and oecircFNTA can increase FNTA expression, and that this was more profound than using oelinearFNTA in J82 cells (lower panel).

(B) A RNA pull-down assay was used to test the binding of circFNTA to 11 miRNAs (predicted by online database) in UMUC3 cells. The results showed that miR-328-5p, miR-370-3p and miR-920 can be pulled-down by circFNTA.

(C) Relative miRNA expressions were quantified by qRT-PCR to test miRNA overexpression efficacy.

(D-E) Transwell invasion assay (D) confirmed that only oemiR-370-3p can reverse oecircFNTA induced invasion in J82 cells, and MTT assays (E) confirmed that oemiR-370-3p can reverse oecircFNTA induced cisplatin chemo-resistance (2 μg/ml) in J82 cells. Scale bar, 100 μm.

(F-G) Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Western blot (F) and Transwell assay (G). Scale bar, 100 μm.

(H) MTT assays also confirmed that transducing with a miR-370-3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo-resistance (2 μg/ml) reduction by shcircFNTA.

(I) The structure of the circFNTA (WT and Mut) binding sites of miR-370-3p. Mut and WT circFNTA's were constructed based on the miRNA target sites.

(J) The transfection efficacy of oecircFNTA WT and oecircFNTA Mut was tested by qRT-PCR.

(K) Transfection with oecircFNTA WT could increase J82 cells invasion, while oecircFNTA Mut cannot. Scale bar, 100 μm.

(L) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo-resistance (2 μg/ml), while oecircFNTA Mut did not have this effect.

(M) The structures of target sites of miR370-3p at the FNTA 3'UTR.

(N) Luciferase reporter activity after transfection of WT and Mut FNTA 3'UTR reporter constructs into UMUC3 cells (left panel), comparing transduced miR-370-3p inhibitor vs Ctrl and J82 cells (right panel), and comparing oemiR-370-3p vs control PLV.

Data information: (B, C, D, E, G, H, J, K, L and N) Data shown represent the means ± SD (n = 3 biological replicates). Student's t-test was used for the statistical analysis (*P < 0.05, **P < 0.01, ***P < 0.001, NS=Not significant).
Androgen receptor-regulated circFNTA activates KRAS signaling to promote bladder cancer invasion (2020)
Figure 5

Figure 5. circFNTA modulates FNTA to promote BCa invasion and chemo-resistence by activating KRAS.

(A) Western blots show the knock-down efficiency of shFNTA in J82 and UMUC3 cells.

(B) Transwell assays indicated that shFNTA can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Quantifications are shown on the right. Scale bar, 100 μm.

(C) MTT assays also confirmed that shFNTA can reverse oecircFNTA-increased chemo-resistance to cisplatin (2 μg/ml) in J82 and UMUC3 cells.

(D-E) Transwell assays (D, with quantitations at the right) and MTT assays (E) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.

(F) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 cells did not change KRAS protein level significantly.

(G) Raf-1 RBD GST-pull-down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 cells (upper panel), and oecircFNTA can increase KRAS activity in J82 cells (lower panel).

(H) Western blots showed that shcircFNTA can decrease ERK1/2 and MEK1/2 phosphorylation in UMUC3 cells (left panel), and oecircFNTA can increase their phosphorylation in J82 cells (right panel).

Data information: (B, C, D and E) Data shown represent the means ± SD (n = 3 biological replicates). Student's t-test was used for the statistical analysis (**P < 0.01, ***P < 0.001, NS=Not significant).
Androgen receptor-regulated circFNTA activates KRAS signaling to promote bladder cancer invasion (2020)
Figure 6

Figure 6. Preclinical study using in vivo mouse model to confirm the role of circFNTA in bladder cancer cell progression and cisplatin chemoresistance.

(A) UMUC3-PLKO and UMUC3-shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model, and cisplatin (1 mg/kg) was intraperitoneally administered. Images of tumors are shown after mice were sacrificed (N= 6).

(B) Compared with the vector (PLKO) group, the tumor growth rate was significantly inhibited in shcircFNTA nude mice.

(C) Tumor weights also decreased in the shcircFNTA group.

(D-E) The qRT-PCR results using mouse tumor tissues revealed that circFNTA expression was significantly reduced (D), and the expression of its host gene FNTA was correspondently decreased in the shcircFNTA group as compared to the PLKO group (E).

(F) Representative immunohistochemistry (IHC) images detecting FNTA, p-ERK1/2 and p-MEK1/2 in PLKO tumurs as compared to shcircFNTA xenografted tumors. Scale bars: 100 μm (upper panels) and 25 μm (lower panels).

(G) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA, p-ERK1/2 and p-MEK1/2.

(H) The tumor tail vein injection model was established to observe lung metastasis (N=6). Representative images of lung metastasis foci in gross and microscopic HE examination. Scale bars: 400 μm (upper panels) and 100 μm (lower panels).

(I) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group.

(J) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group.

Data information: (B, C, D, E, G and J) Data shown represent the means ± SD (6 biological replicates). Student's t-test was used for the statistical analysis (*P < 0.05, **P < 0.01, ***P < 0.001).
Suboptimal resource allocation in changing environments constrains response and growth in bacteria (2021)
Figure 1

Fig. 1: Diauxic shift from glucose to acetate.

(A) Diauxic growth of WT E. coli (NCM3722) in minimal media containing glucose and acetate, bacterial density measured as optical density (OD600). Growth on glucose is captured by the exponential fit (blue dashed line), and proceeds until glucose runs out (black dashed line) at time = 0 h. This is followed by a period of growth-lag lasting ~3.5h before exponential growth resumes on acetate (red dashed line).

(B) The central carbon metabolism pathways are illustrated along with the nodes branching out into amino acid precursor synthesis (green arrows). Glucose to acetate diauxie requires switching from the pathways facilitating glucose consumption (represented in blue) to those responsible for acetate consumption (represented in red). More details in Fig. EV1B.

(C) The mRNA fractional abundances for aceB, aceA and the gluconeogenesis genes (summed abundance of maeA, maeB, pck, and ppsA genes) were estimated by RNA sequencing performed at various time points during the diauxic transition as indicated on the x-axis. The x-axis is truncated 2.3 hours into the shift and the "post shift" (pink) regime represents transcript levels when growth fully resumes on acetate, measured under steady state growth on acetate minimal medium. The series of RNA-seq through the growth transition was performed once.

(D) Lag-times for controlled titration of aceBA expression using an inducer construct in strain NQ1350 (inset). Addition of chlorotetracycline (cTc) removes the tetR repression and induces aceBA expression. As the expression of aceB/aceA during the response (inducer added at time = 0h) is increased, lag times decrease. Bar plot shows mean lag-times (N=3 biological repeats) for different inducer concentration with error bars denoting the Standard Deviations (SD).

(E) Of the overall transcription and translation fluxes (arrows), a strong allocation towards the expression of shunt and gluconeogenesis genes (red) increases the novel synthesis of required enzymes and should thus lead to faster growth recovery.
Suboptimal resource allocation in changing environments constrains response and growth in bacteria (2021)
Figure 4

Fig. 4: Expression of non-required genes delay growth-recovery during the glucose acetate shift.

(A) The major gene categories up-regulated in balanced growth on acetate (red) compared to that on glucose (blue). mRNA abundances determined by RNA-Seq are represented as percent of total mRNA in the given growth condition. Genes are categorized using the COG classification(Tatusov et al, 2000) and porins were classified by manual curation using annotations available from ecocyc.org.

(B) For the various gene categories in panel A, the temporal kinetics of expression during the diauxic shift is plotted. Time 0 indicates time when glucose runs out. The series of RNA-seq through the growth transition was performed once.

(C) Steady-state growth in glucose and acetate for the WT and the motility deletion strains ΔfliC and Δflh. Growth rate differences between WT and the mutants are not significant in glucose (p-value >0.05), but are significant for growth in acetate (p-values<0.02). Additional growth conditions are shown in Fig. EV6.

(D, E) Growth transitions for the motility deletion strains ΔfliC and ΔflhD are substantially faster than that for the WT strain, with (D) showing representative growth kinetics of the three strains and (E) showing mean lag-times and standard error of four independent biological replicates. Lag-times for WT are significantly different from that of ∆fliC (p-value 0.01) and ∆flhD (p-value 0.004).

(F) ∆fliC shows a 2-fold increase (p-value 0.17) and ∆flhD shows a 3-fold increase (p-value 0.02) in aceB expression compared to WT, as measured by qPCR.

Data information: Mean of N=(4,4,2) biological repeats is shown in (C,E,F) respectively, with error bars denoting SD. All indicated p-values were computed by a two-sample t-test.
Suboptimal resource allocation in changing environments constrains response and growth in bacteria (2021)
Figure 5

Fig. 5: Expression of non-required genes is responsible for lags during many diauxic shifts.

(A) The expression of different uptake and metabolic proteins are required for the utilization of different carbon sources. Points of entry into the central metabolism are indicated.

(B-D) Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT (black) and the motility deletion strains ΔfliC and ΔflhD strain (magenta and green).

(E) Derived lag-times for the different growth transitions. Means based on N=4 biological repeats are shown. Error bars denote SD. and lag-time difference between WT and the two mutants are significant for shift to each transition (p-value<0.05; two-sample t-test).

Expanded view figure legends
Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient (2020)
Figure 3.

Cell lines susceptible to SARS-CoV-2. (A-C) Cell lines were infected with a high MOI of SARS-CoV-2 (MOI >5), washed after adsorption, and subsequently harvested 24 hours post infection for viral titer and protein lysates. A) Viral titer for SARS-CoV-2 was quantitated by plaque assay on Vero E6 cells two days post inoculation. B & C) Protein lysates were probed by western blot with B) anti-SARS nucleocapsid (N) antibody or C) anti- SARS-CoV spike (S) antibody.
SARS-CoV-2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage (2021)
Figure 1

Figure 1. IL-1β secretion is impaired in SARS-CoV-2 infected monocytes.

(A-C) Reanalysis of published single-cell sequencing data (GSE163668) of SARS-CoV-2 patient PBMCs. t-Distributed Stochastic Neighbor Embedding (tSNE)-based visualization of single-cells containing PBMCs from healthy donors, SARS-CoV-2 infected mild/moderate and severe patients was shown on the left panel and cluster annotations were shown on the right (A). Percentage of non-classical monocytes in each sample group were calculated (B). Relative average expression levels of IL1B and NLRP3 in non-classical monocytes of each group were calculated (C). n=20 for healthy donor, n=9 for mild/moderate, n=7 for severe. For B, C, each dot represents an individual. For B, C, Brown-Forsythe and Welch ANOVA test was used and data were shown as means±SD. *, P<0.05; **, P<0.01; NS, non-significant.

(D-F) CD14+ monocytes sorted from healthy PBMCs were infected with SARS-CoV-2 viruses at an MOI of 1 for 1 h, followed by washing away extracellular viruses and further culture to meet the indicated infection time. Supernatants were collected and subjected to RT-PCR analysis of viral RNAs (D) and ELISA assay of IL-1β (E). The percentage of cells undergoing cell death was calculated through checking released LDH in the supernatants (F). For D-F, Student's t-test was used and data were shown as means±SD of three technical replicates. ***, P<0.001; results were repeated for three times with similar results.

(G) Sorted human monocytes were incubated with control medium or SARS-CoV-2 viruses at an MOI of 1 for 1 h, followed by washing away extracellular viruses and further culture for 1 h. Cells were fixed and stained with the indicated antibodies. Otherwise, monocytes were challenged with 1 μg/ml LPS for 3 h and 10 μM nigericin for 30 min, followed by fixing and staining with the indicated antibodies. Treatment with medium from uninfected Vero cells served as a control. Scale bar: 5 μm.

(H-J) Supernatants from control or SARS-CoV-2 infected CD14+ monocytes with or without LPS and nigericin stimulation were collected and subjected to an ELISA assay for IL-1β levels (H). Cell viability in cell pellets were examined using a cell viability assay through checking ATP levels inside cells (I). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (J). For H-J, Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results.

Data information: For B, C, Brown-Forsythe and Welch ANOVA test was used and data were shown as means±SD. For D-F and H-J, Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. For D-J, results were repeated for three times with similar results.
SARS-CoV-2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage (2021)
Figure 2

Figure 2. IL-1β secretion and pyroptosis are inhibited by SARS-CoV-2 nucleocapsid in human monocytes.

(A) NLRP3+/+ and NLRP3-/- THP-1 cells were incubated with SARS-CoV-2 at an MOI of 1 for 1 h, followed by washing away extracellular viruses and further culture for 3 h. Otherwise, cells were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cell pellets were collected and immunoblotted with antibodies against the indicated proteins. Treatment with 1 μg/ml LPS for 3 h served as a control.

(B, C) THP-1 cells stably expressing the indicated SARS-CoV-2 genes were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were collected for an ELISA assay to determine supernatant IL-1β levels (B) and cell pellets were subjected to a cell viability assay to verify the percentage of viable cells through checking cellular ATP levels (C). For B-C, Student's t-test was used and data were shown as means±SD of three technical replicates. NS, non-significant.

(D-F) THP-1 cells stably expressing the nucleocapsid gene or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants and cell pellets were collected and immunoblotted with antibodies against the indicated proteins (D). The percentage of viable cells was determined through checking cellular ATP levels in cell pellets (E). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant.

(G-I) Human CD14+ monocytes were electroporated with plasmids encoding the nucleocapsid or control vector for 36 h, followed by stimulation with 10 μM nigericin for 30 min post pretreatment with 1 μg/ml LPS for 3 h. Supernatants and cell pellets were collected and immunoblotted with antibodies against the indicated proteins (G). The percentage of viable cells was determined through checking cellular ATP levels in cell pellets (H). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (I). For H, I, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant.

Data information: For B-I, control vector used here was empty vector corresponded to the nucleocapsid-containing vector. For B, C, E, F, H, I, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Similar results were observed for at least three times.
SARS-CoV-2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage (2021)
Figure 4

Figure 4. SARS-CoV-2 nucleocapsid protects GSDMD from cleavage by caspase-1. (A, B) THP-1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (A) or stained with propidium iodide (PI) (B, left). Scale bar: 20 μm. The percentage of PI positive cells was calculated (B, right). Student's t-test was used and data were shown as means±SD of three technical replicates. ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results.

(C, D) Sorted human CD14+ monocytes were electroporated with plasmids encoding nucleocapsid or control vector for 36 h and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were immunoblotted with antibodies against the indicated proteins (C). Otherwise, cells were stained with PI and the percentage of PI positive cells was calculated (D). Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Results were repeated for three times with similar results.

(E, F) GSDMD+/+ and GSDMD-/- THP-1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were collected for an ELISA assay to determine IL-1β levels (E). Cell pellets were subjected to a cell viability assay through checking cellular ATP levels (F). For E-F, Student's t-test was used. Data were shown as means±SD of three technical replicates. NS, non-significant. Results were repeated for three times with similar results.

(G) An in vitro cleavage assay was performed by incubating GSDMD and caspase-1 p20/p10 complex with or without the presence of nucleocapsid, followed by immunoblotting with antibodies against the indicated protein tags. FLAG-GSDMD was immunoprecipitated from HEK293T cells using anti-FLAG coupled magnetic beads. His-tagged p20, p10 and nucleocapsid were purified from E. coli strain BL21.

(H, I) THP-1 cells stably expressing full-length nucleocapsid or mutant lacking amino acids 70-160 and 290-360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (H). Cells were also stained with PI and the percentage of PI positive cells was calculated (I). Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Results were repeated for three times with similar results.

Data information: For A-F, H, I, control vector used here was empty vector corresponded to the nucleocapsid-containing vector. For B, D-F, I, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.
SARS-CoV-2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage (2021)
Figure 5

Figure 5. SARS-CoV-2 nucleocapsid protects GSDMD linker region from caspase-1 mediated cleavage.

(A) GST or GST-GSDMD, p20 (C285A)-His, p10-His and increasing amount of His-nucleocapsid were incubated for a GST-pulldown assay, followed by immunoblotting with antibodies against the indicated protein tags.

(B) An in vitro cleavage assay was performed through mixing GSDMD and its mutants with caspase-1 p20/p10 complex, followed by immunoblotting with antibodies against the indicated protein tags. β-actin served as a loading control. FLAG-tagged GSDMD and its mutants were immunoprecipitated from HEK293T cells using anti-FLAG coupled magnetic beads. HA-tagged p20 and p10 were immunoprecipitated from HEK293T cells using anti-HA coupled agarose beads.

(C) A GST-pulldown assay was performed through incubating GST-tagged GSDMD and mutants with His-tagged nucleocapsid, followed by immunoblotting with antibodies against the indicated protein tags. GST-GSDMD, GST-GSDMD mutants and His-nucleocapsid were purified from E. coli strain BL21.

(D) GST-GSDMD and its mutant lacking amino acids 265-284 were mixed with His-tagged nucleocapsid, followed by a GST-pulldown assay. Input and precipitates were immunoblotted with antibodies against the indicated protein tags.

(E) GST-tagged GSDMD linker containing amino acids 270-280, 265-284 or 260-290 were incubated with His-tagged nucleocapsid, followed by a GST-pulldown assay visualized with Coomassie blue staining.

(F) Schemes for GSDMD linker substitution and GSDMD chimera. GSDMD linker spanning amino acids 265-284 were replaced by a mCherry sequence except for the amino acid D275 (GSDMDsusti., upper panel). mCherry-GSDMD-C chimera was generated by displacing GSDMD N-terminus with mCherry (lower panel). The linker was also changed as above.

(G, H) Wild-type GSDMD and GSDMDsusti. (G) or mCherry-GSDMD-C (H) were purified from HEK293T cells and incubated with recombinant p20/p10 complex and His-tagged nucleocapsid in a cleavage buffer, followed by immunoblotting with antibodies against the indicated protein tags or antibody against GSDMD C-termimus.

(I) Plasmids encoding wild-type GSDMD, GSDMDsusti. or mCherry-GSDMD-C were co-transfected with nucleocapsid into HEK293T cells, followed by immunoprecipitation with antibody against FLAG. Input and precipitates were immunoblotted with antibodies against the indicated protein tags.

Data information: Experiments were repeated for three times with similar results.