EMBO/soda-nel huggingface dataset:
Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis (2016)
Figure 2

Figure 2: CLU modulates Cdc25 expression in vitro.

(A) Real-time PCR for CLU and Cdc25C in three different prostate cancer cell lines. Target genes expression was calculated relative to GAPDH and normalized to siSCR. Error bars represent mean ± SD, n=3. (B) Western blot for CLU and Cdc25C after siSCR or si CLU transfection in the same prostate cancer cell lines as in A. Vinculin was used as loading control. (C) Western blot for CLU and Cdc25C in PC3 cells after transfection with siSCR or siCLU followed by transfection with CLU or empty expression plasmid for additional 48h. Vinculin was used as loading control
Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis (2016)
Figure 3

Figure 3: Clusterin and Cdc25C are inversely correlated in vivo and in human patients.

(A) Upper panel: Western blot for CLU and Cdc25C from LNCaP xenografts harvested from mice treated with OGX-011 (n=8) or SCR ASO control (n=6). Vinculin was used as loading control. Lower panel: Quantification of the protein levels, relative to the loading control, reported as dot plots. *p<0.05 by t-test followed by Welch’s correction (CLU p= 0.049, Cdc25C p= 0.024). (B) Western blot with the correspondent quantification of protein levels (left) and real time PCR (right) from untreated LNCaP xenografts harvested at endpoint. The negative correlation between CLU-RNA and Cdc25C-RNA ΔΔCT values was calculated using a spearman test (rho=-0.86; p=0.0107). (C) Immunoistochemical staining for CLU and Cdc25C of radical prostatectomy samples from historical control specimens with no neoadjuvant therapy (n=5) or neoadjuvant hormone therapy, (NHT) (n=5) or the combination of NHT and OGX-011 (n=9) for 3 months prior surgery. Average score staining from the 3 groups (left) and representative sections for CLU and Cdc25C (right). Error bars represent mean ± SEM. CLU: *p<0.05; untreated versus 3mNHT (p=0.0213); 3mNHT versus 3mNHT +OGX-011 (p=0.012) by ANOVA followed by Bonferroni post hoc analysis. All cases were normalized by clinical stage, Gleason score and serum PSA. For each patient, the pathologist selected the area with the highest Gleason score. Scale bar represents 100 µm
Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis (2016)
Figure 4

Figure 4: Analysis of Cdc25C/CLU complex in PC3 cells by co-immunoprecipitation, immunofluorescence microscopy and in situ proximity ligation assay (PLA)

(A) Co-immunoprecipitation from parental PC3 cells transfected with CLU and Cdc25C plasmid expression vectors. Proteins were immunoprecipitated with specific anti-Cdc25C (left) and CLU (right) antibodies and analyzed by immunoblotting. (B) Dual immunofluorescence staining with Cdc25C (green) and CLU (red) antibodies in PC3 cells. Confocal microscopy was used to identify the interaction (white arrows). DNA was counterstained with (DAPI) (blue). Scale bar represent 10 µm. (C) Duolink proximity ligation assay between Cdc25C and CLU in PC3 cells. Confocal microscopy was used to detect the interaction (red dots). DNA was counterstained with DAPI (blue). PC3 cells transfected with siCLU were used as a negative control. Scale bar represent 10 µm.
Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis (2016)
Figure 6

Figure 6: Decreased PP2A activity causes Cdc25-T48 accumulation after CLU silencing

(A) Left panel: Western blot for CLU, Cdc25C, Cdc25C-T48, Cdc25C S216 and Cdc25C S198 in PC-3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Vinculin was used as loading control. Right panel: Immunofluorescence for Cdc25C-T48 in PC3 cells after siSCR or siCLU transfection. DNA was counterstained with (DAPI) (blue). Scale bar represents 10 µm.(B) Western blot for CLU, Cdc25C-T48, Cdk1 and PP2A in PC3 cells after siSCR or siCLU transfection and after synchronization followed by treatment with or without okadaic acid (OA) (100 nM) for 12 hours. Proteins were extracted 15 min after nocodazole release. Vinculin was used as loading control. (C) Western blot for PP2A, CLU, Cdc25C-T48, Cdc25C, Wee1 and Cdk1 in PC3 cells transfected with siSCR or siPP2A synchronized and nocozodole released at indicated time points. Vinculin was used as loading control. (D) PP2A phosphatase activity measurement in PC3 cells after siSCR or siCLU transfection, synchronization and nocozodole release at indicated time points. Error bar represent mean ± SEM, n=3, ** p<0.01 (p=0.0025) by ANOVA followed by Bonferroni’s post hoc comparisons. Cells treated with no peptide were used as a negative control.
Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis (2016)
Figure 7

Figure 7: Up-regulation of Wee1 kinase compensates constitutive Cdk1 and Cdc25C activation in G2/M phase after CLU silencing.

(A) Western blot for CLU, Cdk1 Y15, Cdk1 and Wee1 in PC-3 cells after siSCR or siCLU transfection followed by synchronization and nocozodole release at indicated time points. Vinculin was used as loading control (B) Wee1 expression level was evaluated in PC3 cells after CLU silencing by real-time PCR. Error bar represent mean ± SEM, n=3, *** p<0.001 (p=0.0001) by unpaired t-test followed by Welch’s correction. (C) FACS analysis showing percentage of the PC3 cells positive for phosphohistone H3 after transfection with siSCR or siCLU in presence and absence of Wee1 inhibitor, MK-1775. Error bar represent mean ± SEM, n=3, ****p<0.0001 by Mann-Whitney (D) Western blot for CLU, Wee1, Cdk1 Y15, Cdk1 and Cyclin B1 in PC3 cells after transfection with siSCR or siCLU followed by synchronization and treatment with or without MK-1775, a specific Wee1 inhibitor, for 12 hours. Cells were harvested at different time points after nocodazole release. Vinculin was used as loading control.
Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis (2016)
Figure 8

FIGURE 8: CLU silencing increases cabazitaxel efficacy.

(A) Cabazitaxel IC50 calculation using WST1 assay in PC3 cells after transfection with siSCR or siCLU (left), and SCR or OGX-011 (right) followed by 72h of cabazitaxel treatment. Percentage of surviving cells was calculated relative to control. Error bar represent mean ± SEM, n=3. Inset: Western blot was performed to verify CLU knockdown in cells. (B) Mitotic cells and cells in mitotic catastrophe were counted using DAPI in PC-3 cells after SCR or OGX-011 transfection followed by different concentration of cabazitaxel as indicated (>1000 counts) Error bar represent mean ± SEM.,*p<0.05, ***p<0.001, ****p<0.0001 by Student's t-test.( caba 1.5 nM p=0.00115 ; 3 nM p=0.0105 ; 4.5 nM p=1.93x10-7, ; 6.25 nM p=8.9x10-9; 12.5 nM p=6.8x10-9; 25 nM p=1.2x10-9) (C) Clonogenic assay in PC-3 cells after SCR or OGX-011 transfection followed by treatment with different concentrations of cabazitaxel as indicated. The number of survival clones was assessed after 15 days using crystal violet assay. Error bars represent mean ± SEM n=3,*p<0.05, **p<0.01 by Mann-Whitney (0 nM caba p=0.039; 0.5 nM p=0.0079; 2 nM p=0.0079)
Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis (2016)
Figure 9

FIGURE 9: CLU is required for rapid mitotic exit in cabazitaxel-resistant PC3 cells

(A) Cell cycle distribution of syncronized PC3 and PC3-R-caba cells 60 min after nocozadole release at indicated time points. (B) Western blot for CLU, Cdc25C, Cdc25C-T48, Cdk1, Cdk1-Y15 and Wee1 in IGRCaP-1 and PC3 parental (WT) or cabazitaxel-resistant (R-caba) cells as well as for PC3-R-caba cells transfected with siSCR or siCLU. Vinculin was used as loading control. (C) Cell survival measured using WST1 in PC3-R-caba cells after SCR or OGX-011 transfection and treatment with 10 nM cabazitaxel for 48h. Error bar represent mean ± SEM, n=3, *** p<0.001 by one way ANOVA followed by Bonferroni‘s post hoc correction. (D) Cell survival measured using WST1 in PC3 resistant cells after transfection with siSCR or siCLU followed by treatment with cabazitaxel (10 nM; C10), MK-1775 (10 nM; MK10) or both for 72 hours. .Error bar represent the mean ± SEM, n=3, *** p<0.001 by one way ANOVA followed by Bonferroni‘s post hoc correction. (E) PARP cleavage was assessed by western blot in cells treated as in D and its relative expression was quantified by densitometry using ImageJ. Vinculin was used as loading control (F) Left panel: Quantification of tumour volume over 52 days in nude male mice xenotransplanted with PC3-docetaxel resistant cells and treated with either MK-1775 (M, black line), MK-1775 and micellar taxane (MT, blue line), micellar taxane and OGX-011 (TO, red line) or MK-1775, micellar taxane and OGX-011 (MTO, green line). The error bars indicate mean ± SEM, n=8. Right panel: Slopes of tumor growth between groups, were compared by ANOVA followed by Dunnett‘s post hoc analysis..*p<0.05, ***p<0.001 .p=0.0488 (M to TO); p=0.0007 (M to MTO).
Therapeutic development of group B Streptococcus meningitis by targeting a host cell signaling network involving EGFR (2020)
Figure 2

Fig. 2. Group B Streptococcus (GBS) exploits host S1P2 for penetration of the blood-brain barrier.

(A) Tyrosine phosphorylation of EGFR in HBMEC pre-treated with 20 μM S1P2 antagonist (JTE-013) or vehicle control and infected with GBS strain K79 infection for 60 min. EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79.

(B) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors. All the inhibitors were used at 10 μM. Data represent the means ± SEM from three independent experiments, with statistical analysis by Student's t-test. From left to right, p value of DJB-V-39 is 0.0047, p value of SKI is 0.0062, p value of FTY720 is 0.0022, and p value of HB56 is 0.0045.

(C) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR/Cas9 (left panel). Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC. Data represent the means ± SEM from three independent experiments, with statistical analysis by Student's t-test. From left to right, p value of K79 is 0.0018, p value of K160 is 0.0091, p value of K161 is 0.0054, p value of K181 is 0.00040, p value of K226 is 0.00032, p value of K237 is 0.040, p value of K238 is 0.041 and p value of P539 is 0.0025.

(D) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control. Data represent the means ± SEM from three independent experiments, with statistical analysis by Student's t-test, **p = 0.00084.

(E) Bacterial counts recovered from the blood and brain of wild type (n = 5) and S1P2-/- mice (n = 5) infected with strain K79 for 1h. Data represent the means ± SEM with statistical analysis by Student's t-test, ***p = 4.6E-05.
Therapeutic development of group B Streptococcus meningitis by targeting a host cell signaling network involving EGFR (2020)
Figure 3

Fig. 3 GBS exploits host EGFR, S1P-S1P2 and cPLA2α-CysLT1-ERM for penetration of the blood-brain barrier.

(A) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC.

(B) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control, EGFR knockout and S1P2 knockout HBMEC.

(C) Bacterial counts recovered from the blood and brain in wild type mice (n = 5) and CysLT2-/- mice (n = 5) infected with strain K79 for 1h. Data represent the means ± SEM with statistical analysis by Student's t-test, p = 0.074.

(D) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA (left panel) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC. Data represent the means ± SEM from three independent experiments, with statistical analysis by Student's t-test. From left to right, p value of K79 is 0.0039, p value of K160 is 0.0082, p value of K161 is 0.00091, p value of K181 is 0.0087, p value of K226 is 0.00020, p value of K237 is 0.0039, p value of K238 is 0.00066, p value of P539 is 1.76E-05 (right panel).

(E) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control. Data represent the means ± SEM from three independent experiments, with statistical analysis by Student's t-test, **p = 0.0021.

(F) Bacterial counts recovered from the blood and brain in wild type mice (n = 7) and CysLT1-/- mice (n = 7) infected with strain K79 for 1h. Data represent ± SEM with statistical analysis by Student's t-test, *p = 0.021.

(G) Ezrin protein expression in ezrin knockdown HBMEC using shRNA (left panel) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC (right panel). Data represent the means ± SEM from three independent experiments, with statistical analysis by Student's t-test, **p = 0.0044.

(H, I) Ezrin phosphorylation in HBMEC pre-treated with CysLT1 antagonist (montelukast) and infected with GBS strain K79 for 60 min (left panel) and ezrin phosphorylation in the homogenates of brain capillaries of CysLT1-/- and control mice infected with GBS strain K79 for 1h (right panel).

(J) Intracellular K79 co-localization with EGFR and ezrin in control and S1P2 knockout HBMEC (as shown by arrows, cyan). Percentage of co-localization was calculated by counting numbers of all GBS and co-localized intracellular GBS from at least three representative fields, and expressing as numbers of co-localized GBS/all GBS x 100. Data represent the means ± SEM of at least 60 GBS from three fields, with statistical analysis by Student's t-test. In left panel, *p = 0.034. In right panel, **p = 0.0048.
Therapeutic development of group B Streptococcus meningitis by targeting a host cell signaling network involving EGFR (2020)
Figure 4

Fig. 4. Use of montelukast as an adjunct to an antibiotic (ceftriaxone) in therapy of experimental GBS meningitis.

(A) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79. Data are presented as a Kaplan-Maier plot with a log rank test used to compare percentage of survival between the groups, CFX group n = 18, and CFX + MONT group n = 10*p = 0.015. (CFX: ceftriaxone; MONT: montelukast).

(B) Nissl staining of the hippocampus CA1, CA2 & CA3 regions (low magnification) and cortex region (higher magnification) (left) and histograms showing the number of Nissl-stained bodies (right) Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value between control group and Ceftriaxone group is 0.00021, p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0.013.

(C-D) Representative immunofluorescence images (left) and quantification (right) of TUNEL bodies, astrocyte (GFAP), microglia (Iba1) in cortex of wild type mice after 7 days of therapy. Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value of TUNEL bodies between CFX group and MONT + CFX group is 0.0012, p value of astrocyte between CFX group and MONT + CFX group is 0.0068, and p value of microglia cells between CFX group and MONT + CFX group is 0.0029.

(E) Y-maze test for spatial learning and memory of different group of wild type mice. The total duration of time spent at novel arm (left) and the percentage of entries to novel arm of Y-maze (right). Data represent the means ± SEM was statistically analyzed by One-way ANOVA, p value of total duration of time spent at novel arm between control group (n = 6) and CFX group (n = 5) is 0.00012, p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.00017; p value of total duration of % of entry to novel arm between control group (n = 6) and CFX group (n = 5) is 0.0102, and p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.0216.

Expanded View Materials
Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV (2019)
Figure 1

Figure 1: Complex I and IV enzymatic deficiencies in ∆4-CYB cells.

(A) The activities (mUnits/g of protein) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and ∆4-CYB cells and normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=4-6 biological replicates). Unpaired Student's t-test **p=0.0100; ***p=0.0002; ****p<0.0001.

(B) Complex I in gel activity assays (IGA) after Blue-native gel electrophoresis (BNGE) of WT and ∆4-CYB samples solubilized either with 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 hours (lighter signals in DDM gels) or were left to continue the reaction for 24 hours to obtain darker signals (DDM and Digitonin gels).

(C) BNGE, Western blot and immunodetection, with anti-NDUFS1 (cI), anti-ATP5A (cV) and anti-SDHB (cII) antibodies, of samples from the WT cybrids and from ∆4-CYB clones E (#17.3E) and B (#17.3B). Clone E was the cell line of choice for the analysis shown in panels (A and B), and all the figures hereafter.
Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV (2019)
Figure 3

Figure 3: Blue-native gel electrophoresis (BNGE) mass spectrometry and immunodetection analysis of cIII2-related proteins.

(A) Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised (see also Figures EV1, and EV2). The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, vs. the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines was determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and ∆4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

(B) Second-dimension BNGE of digitonin-solubilized samples from WT and ∆4-CYB cells, western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles.

(C) Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII2 subunits. The x-axis values were the slice number (1-64) and the y-axis values were the relative peptide intensity. The graph shows the mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0083 (UQCRC2); **p=0.0033 (UQCRFS1); *p=0.0224; n.s.=non-significant.

(D) Complexome profiles of two cIII2 assembly factors (BCS1L and LYRM7 or MZM1L) generated in the same way as in (A). The represented values are the mean ± SEM of the two reciprocal labeling experiments.
Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV (2019)
Figure 4

Figure 4: Proteomic analyses of UQCR10 and CYC1-containing protein associations in ∆4-CYB cells. See also Figure EV3.

(A) SDS-PAGE, Western blot an immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing HA-tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).

(B) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the same cell lines as in (A) solubilized either with digitonin or DDM.

(C) BNGE, Western blot and immunodetection, with the monoclonal (M) anti-UQCRQ antibody (Abcam ab110255), of non-transduced ∆4-CYB and WT cells. The mitoplast samples were solubilized with DDM (See also Figure S1).

(D) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments (1 and 2) and anti-HA immunopurification of ∆4-CYB and WT cells expressing UQCR10HA.

(E) Complexome profiles, generated as in Figure 3, for the proteins found specifically enriched in ∆4-CYB UQCR10HA, according to the SILAC immunopurification experiments shown in (D). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

(F) SDS-PAGE, Western blot and immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing an HA-tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).

(G) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the same cell lines as in (F) solubilized either with digitonin.

(H) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments (A and B) of anti-HA immunopurification of ∆4-CYB and WT cells expressing CYC1HA.
Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV (2019)
Figure 5

Figure 5: Complex I alterations in ∆4-CYB cells.

(A) Complexome profiles of the different cI structural modules. The graphs were generated as in Figure 3 but in this case, the peptide intensity values for the individual subunits belonging to each module (Stroud et al., 2016) were averaged to simplify the analysis (see also Figures EV4 and EV5). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

(B) Complexome profiles of the cI assembly factor NDUFAF2 (see main text for details) in WT (blue) and ∆4-CYB (red) mitochondria (see also Figure EV4 and EV5). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

(C) Quantification of the total area under the curve (calculated as in Figure 3C) in the profiles corresponding to each cI module (top graph). The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules. The plotted values are the mean mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test ***p=0.0006; **p=0.0012 (N-module); **p=0.0016 (ND1-module); **p=0.0032 (ND2-module); **p=0.0057 (ND4-module); **p=0.0020 (ND5-module); *p=0.0140.

(D) Quantification of the area of the N-module peak corresponding to the molecular mass of the fully assembled cI: 1,172 kDa in ∆4-CYB samples and the respirasome (cI+cIII2+cIV) SCs in #4.1 WT samples (2,110 kDa). The plotted values are the mean mean ± SD (n=2). Unpaired Student's t-test ****p<0.0001.