EMBO/soda-nel huggingface dataset:
A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain (2020)
Figure 2

Figure 2. High affinity PDZ inhibitors can dissociate PICK1 from transmembrane interaction partner.

Illustration of supported cell membrane sheet (SCMS) assay.

Representative confocal images of SCMS expressing Tac-YFP-DATC24, incubated with pre-complexed fluorescently labelled PICK1 and unlabelled peptides as indicated (10 nM), scale bar 10 µm. Full concentration curves shown in Fig S4 and include the representative images shown here.

SCMS derived competitive binding curves for peptides as indicated, red line indicates 10 nM as shown in representative images.

Experimental outline of pre-incubation with peptide.

Representative confocal images of SCMS expressing Tac-YFP-DATC24, incubated with fluorescently labelled PICK1 and subsequently unlabelled peptide as indicated (10µM). Scale bar 10 µm. Full concentration curves shown in Fig EV2 and include the representative images shown here.

Efficacy of the peptides as indicated to facilitate dissociation of PICK1 from SCMS, red line indicates 10 µM as shown in representative images.

Experimental outline post-incubation with peptide.

Data information: Data points are shown as mean with error-bars as SEM of n ≥ 14. Curves were fitted to a log dose-response (three parameters) extracting the IC50 using GraphPad Prism 7.0. Data points in (F) was compared at different concentrations using two-way ANOVA followed by Tukey's multiple comparisons test (****P<0.0001).
A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain (2020)
Figure 3

Figure 3 Tat-P4(C5)2, but not Tat-C5, reconfigures PICK1 into a novel tetrameric state.

Size exclusion chromatography (SEC) profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of Tat-C5 (purple, Absmax = 11.7 ml)

SEC profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of Tat-P4-(C5)2 (blue, Absmax = 10.7 ml)

SEC profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of TMR-Tat-P4-(C5)2 (blue (abs280 nm), red (abs544 nm), Absmax=10.7 ml).

Representative SEC profiles of PICK1 (40 µM) incubated with increasing concentration of Tat-P4-(C5)2, colors indicate Gaussian fits of respective population, (red) aggregates/Void, (blue) dimeric PICK1, (green) oligomeric PICK1, (orange) unbound Tat-P4-(C5)2.

Quantification of abs280 at Evol = 11.7 ml (dimer - blue) and Evol = 10.7 ml (tetramer - green) as a function of concentration (0-10 µM) of Tat-P4-(C5)2. Light blue line indicates tetramer fraction of total PICK1.

SDS-PAGE sedimentation assay showing increased amounts of PICK1 in the pellet (P) fraction with increasing concentrations of Tat-P4-(C5)2. PICK1~50kDa, Tat-P4-(C5)2~10kDa. S denotes supernatant fraction.

Small angle X-ray scattering (SAXS) derived scattering curves of a concentration range of PICK1 on absolute scale show concentration dependent aggregation.

Pair distance distribution function (pddf) of PICK1 in absence of Tat-P4-(C5)2, indicating very large protein complexes present in sample.

SAXS derived scattering curves of a concentration range of PICK1 in complex with Tat-P4-(C5)2 on absolute scale show minor concentration dependent aggregation.

Pair distance distribution function (pddf) of a concentration range of PICK1 in presence of Tat-P4-(C5)2, indicating a smaller complex than for PICK1 in absence of Tat-P4-(C5)2.

Scattering difference (ΔI(q) = I(q)PICK1+Tat-P4-(C5)2 - I(q)PICK1) between PICK1 in presence and absence of Tat-P4-(C5)2 at indicated concentration shows a clear difference in scattering below 0.1 Å-1.

Proposed EOM ensemble for PICK1, in complex with Tat-P4-(C5)2 (not visible in structure) with shading according to model ensemble percentage (10/20/70 %). C-terminal unstructured regions are removed for clarity.

Data information: In (A), all elution profiles were normalized to the lowest and highest main elution peak. In (D), elution profiles were normalized to the lowest and highest points of PICK1 in absence of Tat-P4-(C5)2. In (G and H) the pddf was fitted using BayesApp. EOM fits were done using known structures of PICK1 PDZ domain (PDB: 2LUI) and the previously published model for the dimeric BAR domain, connected by full flexible linkers, N- and C-terminal. Model in (L) was prepared using PyMoL.
A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain (2020)
Figure 4

Figure 4. Tat-P4-(C5)2 is membrane permeable and compromise PICK1:GluA2 functional interaction in hippocampal neurons

Representative confocal images of hippocampal neurons showing membrane penetration of TMR-Tat-P4-(C5)2 and TMR-Tat-C5 (5µM) (magenta), but not the control TMR-C5 (all 20µM). The cell membrane was stained with DiO (grey). Scale bars: 10µm and 5µm on the zooms/orthogonal views.

Representative confocal images of hippocampal neurons transduced with the viral vector encoding GFP-PICK1 and incubated with 5nM TMR-Tat-P4-(C5)2. Partial co-localization between TMR-Tat-P4-(C5)2 (red), GFP-PICK1 (green) and GluA2 (blue) is seen in insert zooms. Experiment was done three times. Scale bars: 10µm and 1µm on the zooms.

Representative immunoblots from hippocampal slices following treatment with Tat-P4-(C5)2 or Tat-C5 (bath application, 20µM).

Densitometric analysis of immunoblots from C show reduced PICK1:GluA2 Co-IP following incubation with Tat-P4-(C5)2 but not Tat-C5. Bar graphs show mean with error-bars as SEM. (One-way ANOVA followed by Dunnett´s multiple comparisons test, *** p≤0.001, n = 4/group)

Representative immunoblots from hippocampal slices following Tat-P4-(C5)2 bath application (20µM) show reduction in the phosphorylation level of S880 GluA2 both under basal condition and after chemical LTD (cLTD).

Bar graphs show densitometric analysis of immunoblots from (E).

Representative immunoblots from hippocampal slices following Tat-C5 bath application (20µM) show reduction in the phosphorylation level of S880 GluA2 both under basal condition and after chemical LTD (cLTD).

Bar graphs (right panel) show densitometric analysis of immunoblots from (G).

Representative confocal images of hippocampal neurons stained for surface GluA2 (sGluA2, light blue) and internalized GluA2 (iGluA2, red). Treatment with Tat-P4-(C5)2 (20 µM) significantly increases the constitutive, but not PMA induced internalization. Scale bars 10µm.

Quantification of iGluA2/sGluA2 within the region of interest for individual neuron (see I, top right corner). Tat-C5 did not increase GluA2 internalization (see Appendix Fig S9D-F).

Data information: Statistics in (F and H) was done by two-way ANOVA, followed by Sidak post-test, revealed a significant effect of the peptide (F(1, 192) = 27,17), *p ≤ 0.05, **p ≤ 0.01 , n= 4-8 as indicated by individual values. Similar analysis in (J), PMA treatment (F(1, 192) = 217.6),) and a significant interaction factor (F(1, 192) = 40,39), ****p ≤ 0.0001, n=46-50 neurons/group as indicated by individual values. All data expressed as mean with error bars as SEM.
A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain (2020)
Figure 5

Figure 5. Tat-P4-(C5)2 interferes with PICK1:GluA2 functional interaction in mouse spinal cord.

Representative coronal slices from lumbar spinal cord show the presence of both TMR-Tat-P4-(C5)2 and TMR-Tat-C5 peptides (magenta) 1 hour after i.t. administration in naïve mice. Scale bar 200µm. DH dorsal horn, VH ventral horn. Insert zooms of merged channels (right panels), scale bar 50µm.

Representative immunoblots of co-immunoprecipited PICK1:GluA2 from spinal cord lumbar tract total lysates demonstrate partial disruption of this interaction 1 hour after 20 µM i.t. injection of Tat-P4-(C5)2. Densitometric analysis of immunoblots indicate a significant effect following Tat-P4-(C5)2 treatment, graph show mean +/- SEM and individual points, unpaired t-test, **p≤0.01, n=4/group

Representative immunoblots of co-immunoprecipited PICK1:GluA2 from spinal cord lumbar tract total lysates demonstrates no disruption of this interaction 1 hour after 20 µM i.t. injection Tat-C5. Densitometric analysis of immunoblots indicate no significant effect of Tat-C5, graph show mean +/- SEM and individual points, unpaired t-test, p=0.10, n=4/group

Representative immunoblots of lumbar spinal cord total lysates exhibit decreased pS880-GluA2 total levels following 20 µM Tat-P4-(C5)2, and Tat-C5 i.t. injections compared to saline injected animals. Densitometric analysis of immunoblot indicates a significant effect only for Tat-P4-(C5)2 (graph show mean +/- SEM and individual points, One-way ANOVA followed by Dunnett´s multiple comparisons test, ** p≤0.01, n=5-6/group as indicated by individual values)

Surface biotinylation of spinal cord slices under basal condition demonstrates unchanged GluA1 and GluA2 surface level upon Tat-P4-(C5)2, Tat-C5 peptides incubation compared to the untreated condition (CTR). Densitometric analysis of immunoblots show that the reduction in mean surface for GluA2 in not significant (graph show mean +/- SEM and individual points, One-way ANOVA followed by Dunnett´s multiple comparisons test, n=5/group as indicated by individual values).

Immunoblot of PICK1 following pull-down from lumbar spinal cord with Biotin-Tat-P4-(C5)2 or Biotin following 20 µM i.t. administration demonstrating in vivo target engagement.
A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain (2020)
Figure 6

Figure 6. Tat-P4-(C5)2, but not Tat-C5, reduce AMPAR surface expression and allodynia in SNI model of neuropathic pain

Diagram of in vivo timeline (D = day).

i.t. administration (20µM) of Tat-P4-(C5)2 significantly reduced ipsilateral paw hypersensitivity of SNI mice in the initiation stage (2 days after injury) of the spared nerve injury model at time points 1 and 3 hours (n = 7)

i.t. administration (20µM) of Tat-C5 did not affect ipsilateral paw hypersensitivity of SNI mice in the initiation stage (2 days after injury) of the spared nerve injury model (n = 8)

Representative western blots (top) for GluA1 and GluA2 following surface biotinylation of spinal cord slices. Densitometric analysis (bottom) of immunoblots. We observed up-regulation of both GluA1 and GluA2 surface level following SNI surgery compared to non-operated animal CTL (unpaired t test GluA1: t(4)=3.372, GluA2: t(4)=3.991,#p≤0.05, n=3/group). Tat-P4-(C5)2 but not Tat-C5 significantly reduced the SNI-induced GluA2 surface expression and shows a strong tendency for GluA1 as well. Representative immunoblot shows unaltered PICK1 total level in the total lysate in the four conditions.

Images of lumbar spinal cord sagittal sections showing the time course of TMR-Tat-P4-(C5)2 (magenta) after i.t. administration (20 µM) in SNI mice showing maximal accumulation in cells after 60 min. Scale bar 1000 µm and 100 µm in Zooms.

Overlay with immunohistochemical staining for the neuronal marker NeuN (yellow) highlights strong neuronal tropism of TMR-Tat-P4-(C5)2 (magenta). Scale bar 100 µm

Dose-dependent Von Frey test in the acute phase (2 days after injury) of the spared nerve injury model following i.t. administration of Tat-P4-(C5)2 (n=8/group) did not affect ipsilateral paw hypersensitivity of SNI mice and lasts up to 4h post injection.

i.t. administration of Tat-P4-(C5)2 (20 µM) at day 14 and 28 after injury in the maintenance phase of SNI model in mice shows significant increase in pain withdrawal threshold up to 3 hours with equal efficacy in male and female mice (compared to time 0 pre-injection, D14 n=8/group/gender, D28 n=6 male, n=7 female).

Data information: In (B and C) Ipsi- and contralateral paw withdrawal thresholds at different time points were compared to time 0 using two-way repeated measures ANOVA followed by Dunnett's multiple comparisons test (*P<0.05, ****P<0.0001). In (D) GluA1/Pan-Cadherin levels were compared using one-way ANOVA followed by Dunnett´s multiple comparisons test, GluA2: F(2,6)=6.256, *p≤0.05; GluA1: F(2,6)=2.843 p=0.096, n=3/group). In (G) Ipsi- and contralateral paw withdrawal thresholds at different time points were compared to time 0 using two-way ANOVA followed by Dunnett's multiple comparisons test (*P<0.05, **P<0.01, ****P<0.0001). All data expressed as mean ± SEM. In (H) Ipsi- and contralateral paw withdrawal thresholds at different time points were compared to time 0 using two-way repeated measures ANOVA followed by Dunnett's multiple comparisons test (*P<0.05, **P<0.01, ****/####P<0.0001).
PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy (2016)
Figure 1

Figure 1. RT and chemotherapy alter PD-L1 expression in PDAC cells.

Flow cytometry of PD-L1 expression in (A) KPC and (B) Pan02 cells following treatment with gemcitabine and RT. Gemcitabine was diluted in DMSO (vehicle) and DMSO was used for the ‘untreated’ control group (mean ± SD, n=3, One-way ANOVA, Bonferroni test). Western blot analysis of indicated proteins in both KPC (C) and Pan02 (D) cells following radiation and chemotherapy ± AG490, a JAK/Stat kinase inhibitor. Actin represents loading control. Flow cytometry of PD-L1 after gemcitabine and RT (as described above) in KPC (E) and Pan02 (F) following Stat1 downregulation by siRNA (mean ± SD, n=3, Student’s t-test). MFI, mean fluorescence index;
PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy (2016)
Figure 2

Figure 2. PD-L1 blockade does not significantly sensitize PDAC to low RT doses (6 Gy or 5x2 Gy; initiated at day 0) in the syngeneic KPC tumor allograft model.

(A) Tumor growth delay was measured in the different groups, as indicated (n=6 mice per group). Anti-PD-L1 was given at days 0 3, 6 and 9 (black arrows). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment (B) Quantitative data on the percentage of gated cells are shown (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).
PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy (2016)
Figure 3

Figure 3. RT combined with blockade of PD-L1 amplify anti-tumor effects in Pan02 tumor allograft.

(A) Tumor growth delay was measured by treating tumor with RT (12 Gy or 5x3 Gy daily; initiated at day 0) and anti-PD-L1 alone or combination of RT (n=6 mice per group). Anti-PD-L1 was given at days 0, 3, 6 and 9 (black arrows). The radiosensitizing effect of anti-PD-L1 after 12 Gy and 5x3 Gy was assessed as shown by the capped lines (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment. (B) Quantitative data on the percentage of gated cells are shown (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).
PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy (2016)
Figure 4

Figure 4. CD8+ T cells are required for efficacy of RT and anti-PD-L1 treatment.

(A) KPC tumor growth delay after treatment with either 12 Gy (day 0), anti-PD-L1 (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0 3, 6 and 9) or their combinations, as indicated. (B) KPC tumor growth delay after treatment with either 5x3 Gy (day 0-4), anti-PD-L1 alone (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0, 3, 6 and 9) or their combinations, as indicated. (C) KPC tumor growth delay after either simultaneous (day 0, 3, 6 and 9; black arrows-upper row) or sequential (day 6, 9, 13 and 16; black arrows-lower row) addition of anti-PD-L1 to RT (day 0). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A-C is also shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiments.
PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy (2016)
Figure 5

Figure 5. Impact of triple combination of gemcitabine-based chemoirradiation with anti-PD-L1 in the KPC tumor allograft.

(A) Mice were treated with either gemcitabine (day 0, 3), anti-PD-L1 (day 4, 7, 10 and 13; black arrows), 12 Gy (day 4) alone or in combinations, as indicated (n=8 mice per group). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test) No weight loss was observed in the in vivo experiment. RT plus anti-PD-L1 (B) reverses T-cell exhaustion and (C) increases CD8:Treg ratio. Bar graphs show quantitation of T cell activation markers and ratio of CD8:Treg ratio in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).
PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy (2016)
Figure 6

Figure 6. Blockade of PD-L1 enhances the anti-metastatic effect of RT. (A) Representative images of livers from the different treatment groups, as indicated. The corresponding liver weights are shown below (n=6 mice per group). (B) Flow cytometry analysis of CD45+CD8+, CD11b+Gr1+ and CD4+CD25+FOXP3+ cell populations in the livers of the mice from the different treatment groups, as indicated (n=5 per group; (means ± SD, n=1, One-way ANOVA, Bonferroni test).
CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation. (2014)
Figure 1.

Bioinformatics analysis of candidate autophagy-regulating genes and identification of PP2A as a complex regulator of autophagy. (A) All analyzed 318 siRNA target genes were categorized into phosphatase families and marked according to the ability of their corresponding siRNAs to regulate EGFP-LC3 accumulation. PIP, phosphatidylinositol phosphatase; PTP, phosphotyrosine phosphatase; DUSP, dual-specificity phosphatase; PPP, (Ser/Thr) phosphoprotein phosphatase. (B) Canonical pathways with significant enrichment of candidate genes were identified by hypergeometric method using the Ingenuity Pathway Analysis software. Blue bars indicate the −log of the p-values (Fisher’s exact test) of the enrichment (left vertical axis). The orange line indicates the percentage of the identified autophagy-regulating genes of all genes in the respective pathway (right vertical axis). The threshold line indicates the position of the P = 0.05. See Table S1 for genes associated with these pathways. AMPK, AMP-activated protein kinase; ERK, extracellular signal–regulated kinase; ROS, reactive oxygen species; ILK, integrin-linked kinase; iCOS, inducible T cell co-stimulator. (C) Schematic representation of the PP2A holoenzyme that is composed of a catalytic subunit (PP2Ac), a scaffold protein (PR65), and a variable regulatory B subunit that interacts with the complex via PR65-α/β HEAT domains (Huntington/elongation/A subunit/TOR). CIP2A interacts with PP2A holoenzyme and modulates its activity toward specific substrates. The gene names for the identified autophagy-regulating candidates and the corresponding proteins (bold) are indicated. (D) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells 56 h after transfection with the indicated siRNAs. When indicated, cells were exposed to 100 nM rapamycin for the last 3 h. RPTOR (raptor) and BECN1 (beclin1) siRNAs served as positive and negative controls, respectively. (E) Autophagic flux was measured as the ratio between luciferase activities in MCF7-RLuc-LC3WT and MCF7-RLuc-LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h. Error bars are SDs for a representative (n = 5) triplicate experiment with a minimum of 4 × 10 randomly chosen areas/sample analyzed (D) or three independent experiments (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with similarly treated control siRNA-transfected samples.
CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation. (2014)
Figure 2.

CIP2A regulates the mTORC1 pathway in a c-Myc– and Akt-independent manner. (A) Representative immunoblots of indicated proteins from whole-cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0–40 min of amino acid starvation [−AA]). (B) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar (siRNA-resistant CIP2A)-encoding lentivirus, transfected with the indicated siRNAs for 53 h, and treated with 100 nM rapamycin (Rapa) for 0–20 min. (C) MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar-encoding lentivirus were transfected with indicated siRNAs for 48 h and analyzed for EGFP-LC3 puncta formation. (D) MCF7-EGFP-LC3 cells transfected with control (−) and CIP2A siRNAs together with an empty vector (−) or MYC-S62A cDNA as indicated were analyzed 56 h later for EGFP-LC3 puncta (left) and expression of indicated proteins (right). (E) Representative immunoblots (left) of indicated proteins from whole-cell lysates of MCF7 cells treated with the control (C), RPTOR (RP), or CIP2A (CIP) siRNAs for 56 h and densitometric quantification of three independent experiments (right). Error bars show SDs for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with Student’s t test as indicated (C and D) or with control siRNA-transfected samples (E). Black lines indicate that intervening lanes have been spliced out.
CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation. (2014)
Figure 3.

CIP2A regulates cell size, proliferation, and survival. (A, top left) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells infected with control or CIP2A lentiviral shRNAs. Representative immunofluorescence images (right) and immunoblots (bottom) are shown. (B) Mean cell diameters of MCF7-EGFP-LC3 cells infected as in A were measured with a cytometer (NucleoCounter NC-3000; Chemometec AS) equipped with Via1-Cassettes (Chemometec AS). (C) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin (Rapa) for 72 h. The viable cells were either visualized after fixation and crystal violet staining (left; representative figure, n = 4) or trypsinized and counted with NucleoCounter NC-3000 equipped with Via1-Cassettes (right). (D) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA, 2 µM MG132, 5 µM siramesine (Sira), or 100 µM etoposide (Etop) for 48 h, and the metabolic activity (cell density) was measured by the MTT assay (left), and cell death was measured by the LDH assay (right). (E) Light microscopic pictures of MCF10A cells at passage 8 after infection with control or CIP2A-encoding lentivirus. (F) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF10A (left) and HEK-293 (right) cells infected as in E and treated with 100 nM rapamycin for the indicated times. Black lines indicate that intervening lanes have been spliced out. Error bars are SDs for three independent experiments (B) or a representative (n ≥ 3) triplicate experiment (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared by Student’s t test with similarly treated control shRNA-transfected cells or as indicated. Bars: (A and E) 20 µm; (C) 1 cm.
CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation. (2014)
Figure 4.

CIP2A expression correlates with mTORC1 activity in primary human breast tumors. TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores (arbitrary units [a.u.]) for duplicate samples were averaged and plotted according to mean CIP2A expression (left). The red lines are the regression lines, and the stippled red lines define the 95% confidence intervals. Statistical analysis of data was performed using NCSS 2007 and Prism version 5. Examples of the staining of breast cancer (a) and normal (b) cores indicated in the P-T389-S6K1/CIP2A plot are shown on the right. rsp, Spearman’s correlation coefficient.