EMBO/soda-nel huggingface dataset:
A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury. (2015)
Figure 2

The GBS pigment toxin/lipid toxin is pro-inflammatory and cytotoxic to primary human macrophages

A, B PBMC-derived macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by ELISA (B).

C, D PBMC-derived macrophages primed with 100 ng/ml LPS for 3 h were incubated with various concentrations of GBS pigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D).

Data information: Data shown are the average of four independent experiments performed in triplicate, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 4, *P = 0.021 for WT versus ΔcovR; *P = 0.01 for ΔcovR versus ΔcovRΔcylE. (B) n = 4, *P = 0.031 for WT versus ΔcovR; *P = 0.036 for ΔcovR versus ΔcovRΔcylE. (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005.
A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury. (2015)
Figure 3

The GBS pigment toxin/lipid toxin is pro-inflammatory and cytotoxic to immortalized THP-1 monocyte-derived macrophages

A, B WT THP-1 macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B).

C, D WT THP-1 macrophages were incubated with various concentrations of GBS pigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D).

Data information: Data from three independent experiments performed in triplicate are shown, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 3, **P = 0.0080 for WT versus ΔcovR; **P = 0.0016 for ΔcovR versus ΔcovRΔcylE. (B) n = 3, *P = 0.03 for WT versus ΔcovR; *P = 0.02 for ΔcovR versus ΔcovRΔcylE. (C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.
A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury. (2015)
Figure 4

The GBS pigment induces NLRP3 inflammasome-dependent cell death in human macrophages

A, B THP-1 macrophages transfected with empty vector, scrambled control, shASC, or shNLRP3 were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B). Data from three independent experiments performed in triplicate are shown.

C, D The shRNA THP-1 macrophages were incubated with various concentrations of GBS pigment or ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release in supernatants was measured by Luminex assay (D). Pigment-mediated cytotoxicity is dependent on the NLRP3 inflammasome components, suggesting that pigment is inducing a programmed cell death. Data shown are the average of at least three independent experiments performed in triplicate.

Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (A) n = 3, for WT: **P = 0.001 (vector versus shNLRP3), **P = 0.0016 (vector versus shASC); for ΔcovR: ***P = 0.0008 (vector versus shNLRP3), ***P = 0.0001 (vector versus shASC), *P = 0.047 (scrambled versus shNLRP3), **P = 0.0054 (scrambled versus shASC). Data obtained from THP-1/vector was not significantly different from THP-1/scrambled, P = 0.18. (B) n = 3, *P = 0.025. Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.89. (C) n = 3, for 4 μM pigment: **P = 0.0063 (vector versus shNLRP3), **P = 0.0035 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3), **P = 0.009 (scrambled versus shASC); for 2 μM pigment: **P = 0.0088 (vector versus shNLRP3), **P = 0.0066 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3 or shASC); for 1 μM pigment: *P = 0.02 (vector versus shNLRP3), *P = 0.01 (vector versus shASC). Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.99. (D) n = 3, ****P < 0.0001, *P = 0.015.
A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury. (2015)
Figure 5

GBS pigment induces membrane permeabilization and K<sup>+</sup> efflux independently of the NLRP3 inflammasome

A, B THP-1 macrophages proficient for NLRP3 (transfected with scrambled control, A) or deficient for NLRP3 (shNLRP3, B) were treated with 1 μM pigment or ΔcylE extract for 20 min, and propidium iodide (PI) was added during the final 10 min. PI uptake was measured by flow cytometry, and data shown are representative of two independent experiments.

C, D Intracellular potassium concentration was measured by ICP-AES. THP-1 macrophages transfected with the scrambled control (C) or shNLRP3 (D) were treated with GBS pigment (1 μM) or an equivalent amount of the ΔcylE extract. At various time points, cells were lysed and intracellular [K+] was measured relative to untreated cells (see bars and left y-axis), and percent cell death was quantified by alamar blue (see squares, dotted connecting lines and right y-axis). Both NLRP3-proficient and NLRP3-deficient macrophages initially lose intracellular K+ due to GBS pigment (compare t = 0 min to t = 30 min), but the NLRP3-deficient cells (shNLRP3) are able to recover, while the scrambled control do not, demonstrating that initial K+ loss occurs independently of NLRP3. Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett's multiple comparison test following ANOVA; all data were compared to control at t = 0, error bars ± SEM. (C) n = 3, ***P = 0.0002, *P = 0.019, **P = 0.0032 for 120 min, **P = 0.0072 for 180 min. (D) n = 3, **P = 0.0043, *P = 0.031. (E) WT THP-1 macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride, and cytotoxicity was measured by alamar blue assay. The addition of potassium chloride is able to protect the macrophages from cytolysis, demonstrating that K+ efflux is essential for this process. Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM (n = 3, ****P < 0.0001, **P = 0.0036).
A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury. (2015)
Figure 6

The GBS hemolytic pigment/lipid toxin induces caspase 1 activation and pyroptosis

THP-1 macrophages were treated with GBS pigment, and caspase 1 activation was measured by flow cytometry using a FLICA reagent. Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3 cell line, demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome. Results are representative of three independent experiments.

WT THP-1 macrophages were treated with the caspase 1 inhibitor Z-YVAD-FMK, the caspase 3/7 inhibitor Z-DEVD-FMK, or DMSO only prior to treatment with the GBS pigment. YVAD is able to significantly decrease cytotoxicity in cells treated with GBS pigment, demonstrating that caspase 1 is required for GBS pigment-mediated cell death, characteristic of pyroptosis, while DEVD had no effect. Data are average of three independent experiments, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA (n = 3, ****P < 0.0001, ***P = 0.0002, *P = 0.014).
A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury. (2015)
Figure 7

The GBS pigment causes fetal injury by NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent mechanisms

Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106–7 CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.

Scheme of pup numbering in utero and injection site between fetuses P1 and P2 is shown.

In utero fetal death in wild-type CD-1 mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test.

H&E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris.

Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test.

Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM).

IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylE mice and is indicated as NS; P = 0.99.
TUMOR SUPPRESSOR PNRC1 BLOCKS rRNA MATURATION BY RECRUITING THE DECAPPING COMPLEX TO THE NUCLEOLUS (2018)
Figure 1

Figure 1: PNRC1 tumor suppressor hinders oncogene-induced hyperproliferation

A. Visual representation of the signature found associated to hemizygously deleted and downregulated genes across multiple cancer types. The image represents the joint distribution of gene expression regulation (x axis, z-score vs normal tissue) and CNA (y-axis, exact segmentation value). Color intensity is proportional to the density value of the distribution.

B. Quantification of PNRC1 expression levels in primary normal (blue circles) and cancer (red circles) samples by Real Time PCR.

C-D. Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a primary healthy colon (C) or a malignant lymphoma (D). DAPI was used to stain cell nuclei. Scale bars:50µm.

E. Proliferation curves of HeLa cells transfected with HRASG12V (R), PNRC1 (P), their combination (RP) or a LacZ control (L). The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each. A one-way ANOVA with Tukey multiple comparison test was performed for 72h dataset; the statistically significant comparisons are as follows: L vs R (p<0.002), L vs P (p<0.02), R vs P (p<0.0002) and R vs RP (p<0.0004)

F. Proliferation curves of MCF7 cells transfected with HRASG12V (R), PNRC1 (P), their combination (RP) or a LacZ control (L). The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each. A one-way ANOVA with Tukey multiple comparison test was performed for 72h dataset; the statistically significant comparisons are as follows: L vs R (p<0.003), L vs P (p<0.0002), L vs RP (p<0.0001), R vs P (p<0.0001) and R vs RP (p<0.0001)

G. Proliferation curves of KRASG12S-mutated A549 cells transfected with PNRC1 or LacZ control. A representative experiment with the average ± SD of 3 technical replicates is shown. An unpaired t-test was performed for 144h dataset (p<0.0003).

H. Proliferation curves of BJ-T cells transduced with a non-targeting or a PNRC1-specific shRNA construct. Results are shown as the average ± SD of 3 biological replicates with 3 technical replicates each. An unpaired t-test was performed for each time point (** p<0.01, *** p<0.005, **** p<0.001).
TUMOR SUPPRESSOR PNRC1 BLOCKS rRNA MATURATION BY RECRUITING THE DECAPPING COMPLEX TO THE NUCLEOLUS (2018)
Figure 2

Figure 2: PNRC1 interferes with nucleolar RNA metabolism

A. Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1 or RFP control. Confocal images of transfected cells pulsed with EU for 16 hours were collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1-transfected cells are indicated with arrowheads.

B-C. Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against NPM1 (B) or UBF1 (C) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm). Two representative cells are shown for each staining. A magnification of the merged channel images is provided for UBF1 staining.

D. Agarose/formaldehyde gel separation of neotranscribed rRNAs collected from LacZ and PNRC1-expressing cells pulsed for 15 minutes with 3H-uridine and chased for the reported time points (minutes). Samples were loaded according to the amount of incorporated 3H, quantified by liquid scintillation. FU was included as a control of a blocked rRNA processing.

E. Ratios between the intensity of 47S pre-rRNA band and bands belonging to 32S or 28S rRNA species relative to panel (D) and measured at 60 minutes after the chase for both LacZ or PNRC1-expressing cells.

F. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1 or GFP control. The average ± SD of 3 biological replicates is shown. Statistical significance was calculated by an unpaired two-tailed t-test (p<0.0001).
TUMOR SUPPRESSOR PNRC1 BLOCKS rRNA MATURATION BY RECRUITING THE DECAPPING COMPLEX TO THE NUCLEOLUS (2018)
Figure 3

Figure 3: PNRC1 interacts with cytoplasmic RNA decapping proteins

A-B. Coomassie blue staining and western blot analysis of the SDS-PAGE gel corresponding to PNRC1 IP. Red arrows represent the gel areas excised both from the IgG and IP lanes for mass spectrometry analysis. Input, unbound and IP fractions were loaded as reported. The protein band corresponding to PNRC1 is indicated.

C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1 with a specific anti-HA antibody in absence or presence of 1mg/ml RNaseA.

D. Co-immunoprecipitation of DCP1α-interacting proteins performed on HA-PNRC1-expressing HeLa cells with a specific anti-DCP1α antibody.

E. Co-immunoprecipitation performed on wild-type HeLa cells with an anti-PNRC1 antibody and probed for DCP1α.

F-G. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1 with a specific anti-HA antibody and probed with antibodies specific for proteins involved in RNA decapping. The white arrow indicates the specific DCP2 band, as reported in the datasheet of the antibody employed.
TUMOR SUPPRESSOR PNRC1 BLOCKS rRNA MATURATION BY RECRUITING THE DECAPPING COMPLEX TO THE NUCLEOLUS (2018)
Figure 4

Figure 4: Re-localization of the RNA decapping machinery upon PNRC1 expression

A. Confocal microscopy images of RFP or RFP-PNRC1-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1-transfected cells are indicated with arrowheads. Scale bar: 10µm.

B. Quantification of cells classified according to the P-body localization of the three stained proteins. The average ± SD of 3 independent experiments is reported.

C. Nucleolar fractionation performed on LacZ or PNRC1-expressing HeLa cells. Total lysates, cytoplasmic (Cyto), nucleoplasmic (Nucl) and nucleolar (No) fractions were collected. UBF1, RNA PolII and MEK2 were used as markers for the nucleolar, non-nucleolar and cytoplasmic fractions, respectively. The electrophoretic mobility of proteins in the nucleoplasmic lanes is slightly diminished due to nuclear lysis buffer composition.

D. Densitometric quantification of DCP1α nucleolar pool obtained from the western blot shown in panel (C) and normalized upon the amount of nucleolar UBF1 and the levels of DCP1α in the input fraction.

E. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing a shRNA-resistant GFP-DCP1α in combination with RFP or RFP-PNRC1 transgenes (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).
TUMOR SUPPRESSOR PNRC1 BLOCKS rRNA MATURATION BY RECRUITING THE DECAPPING COMPLEX TO THE NUCLEOLUS (2018)
Figure 5

Figure 5: W300A mutation impairs PNRC1 interaction with DCP1α and its nucleolar translocation

A. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS with a specific anti-HA antibody and probed with anti-NPM1 and anti DCP1α antibodies.

B-C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1W300A mutant with a specific anti-HA antibody and probed with antibodies specific for PNRC1-interacting proteins. The white arrow indicates the specific DCP2 band.

D. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing an shRNA-resistant GFP-DCP1α in combination with RFP, RFP-PNRC1WT or RFP-PNRC1W300A mutant (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).

E. Confocal microscopy images of RFP, RFP-PNRC1 or RFP-PNRC1W300A-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads. Scale bar: 10µm

F. Quantification of HeLa cells expressing RFP-PNRC1W300A classified according to the P-body localization of the three stained proteins. The average of 3 independent experiments ± SD is reported.

G. Co-immunoprecipitation performed on HeLa cells expressing LacZ control, HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS constructs with a specific anti-DCP1α antibody and probed with anti-PNRC1 and anti-NPM1 antibodies.
TUMOR SUPPRESSOR PNRC1 BLOCKS rRNA MATURATION BY RECRUITING THE DECAPPING COMPLEX TO THE NUCLEOLUS (2018)
Figure 6

Figure 6: PNRC1 reduces DCP1α/DCP2-dependent rRNA processing and snoRNA decapping

A. Confocal images of a Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1W300A pulsed with EU for 16 hours and collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads.

B. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1W300A or GFP control. The average of 3 independent experiments ± SD is reported.

C. Quantification of 5'-ETS RNA levels in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average of 2 independent experiments ± SD is reported.

D-E. Levels of U3 (D) and U8 (E) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR. Values represents the average of two biological replicates with three technical replicates and are expressed as percentages of enrichment compared to the input ± SD. Where indicated, a two-tailed t-test was performed combining the technical and biological replicates (**** p<0.0001, *** p<0.0002, **p<0.002).

F. Levels of U3 snoRNA immunoprecipitated by the anti-m3G cap antibody in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average ± SD of 2 independent experiments with three technical replicates is reported.

G. Western blot analysis of input and Flag-IP fractions of RIP experiment.

H. RNA levels of U3, U8 and U1 co-immunoprecipitating with GFP, PNRC1WT or PNRC1W300A. Results are shown as the average ± SD of two biological replicates.
Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells (2013)
figf1

(a–d) Rotenone (Rot) increased the number of GFP–LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a–c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig. 3f and Supplementary Fig. S1b,c. Veh, vehicle. (e,f) Rotenone increased delivery of MitoTracker Green-stained mitochondria (mt) to LysoTracker Red (LTR)-stained lysosomes, inhibited by siRNA knockdown of ATG7 or LC3 in SH-SY5Y cells. Ctrl, control. Right: RNAi knockdown. (g) Rotenone decreased IMM (COXIV), OMM (TOM40) and matrix (MnSOD) protein expression levels in primary neurons, reversed by bafilomycin (Baf) inhibition of autolysosomal degradation, quantified in Supplementary Fig. S1e. Mean ± s.d. of
n = 7 independent experiments for b,c, and
n = 3 independent experiments for f,g (see Supplementary Table S4 for statistics source data);
*P0.05 versus vehicle control;
†P0.05 versus Rot/control siRNA. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.
Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells (2013)
figf3

(a–e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (a) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647–annexin V binding. (b–d) PLS3 knockdown had no effect on rotenone-induced autophagy (b), but decreased mitophagy in SH-SY5Y cells treated with rotenone (c; 1 μM) or 6-OHDA (d; 120 μM). (d,e) The effects of PLS3 siRNA were recapitulated using a second siRNA #433 (d, Supplementary Fig. S3b,g), and reversed by transfection with an RNAi-resistant mouse PLS3 vector (e). Inset: PLS3 overexpression. (f–i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f), or with mitochondrial protein immunoblotting (g) and densitometry (h,i and Supplementary Fig. S4c). Knockdown efficiencies are shown in Supplementary Figs S3 and S4. Mean ± s.d. of
n = 4 independent experiments for b,c, and
n = 3 independent experiments for a,d–f,h–i (see Supplementary Table S4 for statistics source data);
*P0.05 versus vehicle;
†P0.05 versus toxin-treated control siRNA;
**P0.05 versus Rot/PLS3 siRNA. See Supplementary Fig. S7 for uncropped blots.