EMBO/soda-nel huggingface dataset:
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 7.

DRP-1 K42A protects from autophagy induced by steroid withdrawal and amino acid starvation. (A and B). Quantification of autophagy in MCF-7 cells. MCF-7 cells were transfected with luciferase or FLAG–DRP-1 K42A followed by steroid withdrawal (serum starvation plus 10−6 M tamoxifen treatment) (A) or by serum and amino acid starvation (B) (mean ± SD calculated from triplicates of 100 cells each). These experiments were repeated three times with reproducible results. Proteins extracted from the transfected cells were subjected to Western blot analysis with anti-FLAG and anti–β-tubulin antibodies. (C) Photographs of the MDC-stained steroid withdrawn MCF-7 cells taken from the same experiment shown in A. Transfections: luciferase (1a and 1b) and DRP-1 K42A (2a and 2b) at low (a) and high (b) magnifications.
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 8.

Autophagy induced by steroid withdrawal or by amino acid starvation is insensitive to caspase inhibitors. (A) Photographs of MDC-stained MCF-7 cells. (Top) Steroid withdrawal. Cells were either grown in complete medium (1), steroid depleted (2), or steroid depleted in the presence of the broad caspase inhibitor BD-fmk (100 μM) (3) for 3 d. (Bottom) amino acid starvation. Cells were either grown in complete medium (1–3) or under amino acid starvation condition (4–6) without (1 and 4) or with (2 and 5) the presence of the broad caspase inhibitor BD-fmk (100 μM) or the general inhibitor of autophagy 3-methyladenine (10 mM) (3 and 6) for 3 d. (B) PARP cleavage in MCF-7–starved cells. Proteins extracted from 4-d steroid-depleted (left) or 12-h TNF–α-treated (100 ng/ml) cells (right) were subjected to Western blot analysis with anti-PARP antibodies. The proteins were prepared from the same experiment shown in A. (C) Graph showing the cumulative phase microscopy scores of dead (shrunk and detached) MCF-7 cells in tamoxifen-treated (filled) or nontreated (unfilled) cultures in the presence (▴) or absence (•) of the indicated caspase inhibitors (25 μM).
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 9.

DAPk antisense RNA protects from IFN-γ–induced autophagy of HeLa cells. (A) Quantitation of autophagy in HeLa cells. Cells were either grown in complete medium (1), treated with IFN-γ (1,000 U/ml) (2), or treated with IFN-γ in the presence of the broad caspase inhibitor BD-fmk (100 μM) (3) for 3 d. (B) Photographs of the MDC-stained HeLa cells corresponding to the experiment in A. Treatments: complete medium (1), IFN-γ (1,000 U/ml) (2), and IFN-γ (1,000 U/ml) + BD-fmk (3). (C) PARP cleavage in HeLa IFN-γ–treated cells. Proteins extracted from IFN-γ–treated (left) or TNF-α–treated (100 ng/ml) cells (right) were subjected to Western blot analysis with anti-PARP antibodies. The proteins were prepared from the same experiment shown in A. (D) Transmission electron micrographs of nontreated (top) and IFN-γ–treated (bottom) HeLa cells. Arrows point to autophagic vesicles. (E) DAPk antisense-transfected and control DHFR-transfected polyclonal populations of HeLa cells were incubated with hygromycin B (200 μg/ml) in the presence or absence of IFN-γ (1,000 U/ml) and BD-fmk (50 μM). Detection of autophagic vesicles was performed 2 d later using the MDC dye. Graphs represent mean ± SD of MDC-positive cells calculated from triplicates of 100 cells each.
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 10.

DRP-1 is localized to the lumen of autophagic vesicles. Immunogold staining of 293 cells expressing HA–DRP-1 reveals a specific staining of DRP-1 in the lumen of double membrane autophagosomes containing cytoplasmic material (A and B) in autophagosomes containing organelles and vacuoles (C) and inside the body of autolysosomes containing digested matter (D). The gold particles do not overlap with the phagocytosed organelles in C or the lysosomes in D.
UBTD1 is a mechano-regulator controlling cancer aggressiveness (2019)
Figure 1

Figure 1: UBTD1 regulates cell mechanical properties via RhoA activation

(A-E, G-H) DU145 cells or (F) A549 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (A) The scatter plot shows the apparent Young's moduli obtained by AFM analysis. Bars represent the median values of the distributions. The immunoblot shows UBTD1 siRNA knock-down efficiency. (B) Immunoblots (left) and quantification (right) show levels of GST-ROCK associated GTP-bound RhoA (RhoA-GTP) and total RhoA in lysates (RhoA) in confluent cells. Immunoblot of UBTD1 shows the level of siRNA depletion. (C) Representative confocal images of focal adhesion (paxillin) and actin cytoskeleton (phalloidin) and outlines of focal adhesions using threshold-based image. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. (D) Cell area, number and area of focal adhesion were quantified (ImageJ) using the analysis as shown (C). Sub-confluent DU145 (E) or A549 (F) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel. (G) Confluent DU145 cells were seeded on 12 kPa matrix. Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 12kPa hydrogel. (H) Representative confocal images of YAP and UBTD1 localization on two matrices of different stiffness. DU145 cells were grown on collagen/acrylamide-coated glass coverslips (soft) or directly on glass coverslips (stiff). Nuclei were stained with DAPI (Blue) on the MERGE image. Scale bar=10 µm; n≥3 independent experiments; *P<0.05; **P<0.01; ****P<0.0001; (A) Mann-Whitney test; (B,D) two tailed t-test; (E-G) Bonferroni's multiple comparison test; data are mean ± s.e.m.
UBTD1 is a mechano-regulator controlling cancer aggressiveness (2019)
Figure 2

Figure 2: UBTD1 is associated with β-catenin at the cell-cell adhesion site

(A) Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1 and β-catenin with cell confluency. E-cadherin was used as a positive control. Tubulin was used as a loading control. (B-C) Left, immunofluorescent labelling of UBTD1 and E-cadherin (B) or β-catenin (C) at the cell-cell contact in confluent DU145 cells and analyzed by confocal microscopy. Right, representative histogram of a co-localization profile prepared with RGB profiler plugin of ImageJ. (D) Immunoblots of UBTD1, β-catenin and YAP after subcellular fractionation of confluent DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). EGFR, RhoGDI and Lamin A/C were used as controls for the membrane (M), cytosol (C) and nucleus (N) compartments, respectively. (E) Co-immunoprecipitation between endogenous β-catenin and UBTD1 was performed in confluent DU145 cells. IgG isotype was used as a negative control. (F) Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 association with E-cadherin or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image. (G) Representative confocal images of β-catenin and UBTD1 in DU145 under resting (Ctrl), after calcium chelator treatment (EGTA) and recovery (Rec) conditions. Nuclei were stained with DAPI (Blue) on the MERGE image. (H) Representative confocal images of β-catenin and UBTD1 in DU145 after cell scattering induced by HGF treatment (24 h). Nuclei were stained with DAPI (Blue) on the MERGE image. Scale bar=10 µm. n≥3 independent experiments; ns=non-significant; ****P<0.0001; (F) Bonferroni's multiple comparison test; data are mean ± s.e.m.
UBTD1 is a mechano-regulator controlling cancer aggressiveness (2019)
Figure 3

Figure 3: UBTD1 promotes YAP degradation by the UPS independently of the Hippo signaling pathway

Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1, p-YAP (ser127) and YAP levels with the cell confluency. Tubulin was used as a loading control. (B-E) DU145 cells or (F) A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Immunoblots of p-YAP (ser127), YAP and UBTD1 (left) and quantification of YAP and p-YAP/YAP levels (right) in control or UBTD1-depleted DU145 at low and high cell density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading controls. (C) Immunoblots of the Hippo signaling pathway using phosphorylated (p-ser909LATS and p-thr183MST1/p-thr180MST2) and total LATS, MST at low and high DU145 cells density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin and Rho-GDI were used as loading controls. In (B) and (C), immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels. (D,E) Immunoblots (up) and quantification (down) of YAP levels in confluent (D) DU145 or (E) A549 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control. (F) Immunoblots (up) and quantification (down) showing YAP levels in UBTD1 depleted confluent DU145 (E) or A549 (F) cells treated or not with MG132 for 6h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control. Numbers at the bottom of the western blots represent the ratio between 6h treatment/0h for each condition. n≥3 independent experiments; *P<0.05; (B) Bonferroni's multiple comparison test; data are mean ± s.e.m
UBTD1 is a mechano-regulator controlling cancer aggressiveness (2019)
Figure 4

Figure 4: UBTD1 regulates YAP ubiquitylation by modulating its association with β-TrCP

(A) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. YAP was used as bait. The IgG isotype was used as a negative control. (B) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. UBTD1 was used as bait. The IgG isotype was used as a negative control. (C) Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 associated with YAP of DU145 and A549 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image. (D) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c and UBTD1. UBTD1 was used as bait. IgG isotype was used as a negative control. (E) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c, YAP and β-catenin. DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single). β-TrCP was used as bait. IgG isotype was used as a negative control. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. (F,G) Proximal ligation assay monitoring (F) and quantification (G) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image. (H) Immunoblots show YAP ubiquitylation in HEK cells in different experimental conditions. Cells were transfected, as indicated, with expression vectors of histidine-tagged ubiquitin (His-Ub) together with control siRNA or UBTD1 siRNA. His-Ub crosslinked forms of YAP were purified (IP: His) and the immunoblot of YAP showed YAP ubiquitylation. The immunoblot of YAP (lower panel) was performed in parallel to verify the amounts of YAP protein engaged in the His-Ub purifications. The immunoblot of UBTD1 shows the level of siRNA depletion. Scale bar=10 µm. n≥3 independent experiments; ns=non-significant; ****P<0.0001; (C) two tailed t-test; (G) Bonferroni's multiple comparison test; data are mean ± s.e.m.
UBTD1 is a mechano-regulator controlling cancer aggressiveness (2019)
Figure 5

Figure 5: Loss of UBTD1 increase YAP nuclear translocation and its activity via ROCK2

(A-B) DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (A) Representative wide-field immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells after UBTD1 depletion. Nuclei were stained with DAPI (Blue) on the MERGE image. (B) Quantification of CTGF1, CYR61 and BIRC5 mRNA levels. (C) Immunoblots of YAP in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1 (+). Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Immunoblot of GFP shows the level of UBTD1 transfection. CHC was used as a loading control. (D) Representative confocal immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1. Nuclei were stained with DAPI (Blue) on the MERGE image. (E) Immunoblots of p-YAP and YAP in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Immunoblot of p-YAP reflects the activity of the Hippo pathway. Immunoblot of UBTD1 and LATS show siRNA knock-down efficiency. Actin was used as a loading control. (F-G) Representative confocal immunofluorescence images (F) and quantification (G) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image. (H) Immunoblots of YAP in DU145 control (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Immunoblots of UBTD1, ROCK1 and ROCK2 show the level of siRNA knock-down efficiency. Actin was used as a loading control. (I-J) Representative confocal immunofluorescence images (I) and quantification (J) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image. (K) Quantification of CTGF1, CYR61, UBTD1 and BIRC5 mRNA in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Scale bar=10 µm; n≥3 independent experiments; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; (A) two tailed t-test; (B,D,G,J,K) Bonferroni's multiple comparison test; data are mean ± s.e.m.
UBTD1 is a mechano-regulator controlling cancer aggressiveness (2019)
Figure 6

Figure 6: Loss of UBTD1 is associated with increased prostate cancer cell aggressiveness and poor prognosis for patients

(A-E) DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (A) Quantification of EMT marker genes (vimentin, slug, snail, fibronectin, α-SMA, E-cadherin) and UBTD1. (B) Immunoblots of EMT markers (vimentin, E-cadherin, slug). Immunoblot of UBTD1 shows siRNA knock-down efficiency. Hsp90 and tubulin were used as loading controls. (C) Quantification of DU145 cell migration in Transwell chamber inserts. (D) Quantification of DU145 cell invasion in Transwell chamber inserts. (E) Invasive properties of human prostate tumoroids depleted for UBTD1. (F) Quantification of UBTD1 mRNA levels in prostate tumor (n=70) and adjacent normal tissues (n=47). (G) Representative UBTD1 immunohistochemistry staining on sections of human prostate tumor and normal tissues (n=8). (H) Representative UBTD1 immunohistochemistry staining on sections of human lung tumor and normal tissues (n=6). (I) The levels of UBTD1 mRNA in grade 3 prostate adenocarcinoma patients (56 patients) are correlated with overall survival (OS). OS was calculated from patient subgroups with mRNA levels that were less or greater than the first quartile value. (J) The levels of UBTD1 mRNA in lung adenocarcinoma patients (522 patients) are correlated with OS. Scale bar = 100µm. n≥3 independent experiments; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; (A, C,D) Bonferroni's multiple comparison test; (E,F) two tailed t-test; data are mean ± s.e.m.
Phosphodiesterase 4A confers resistance to PGE2-mediated suppression in CD25+/CD54+ NK cells (2020)
Figure 1

Figure 1. Expression of PDE4A renders human NK cells less susceptible to PGE2-mediated suppression

A-C) Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for (A) proliferation by flow cytometry for Ki-67 staining after four days of culture (n=6, biological replicates), (B) ability to kill K562 cells after two days of culture in a 4-hour chromium-assay at an E:T ratio of 5:1 (n=4, biological replicates). (C) Viability by flow cytometry using fixable live/dead staining after two days of culture (n=7, biological replicates).

D) Intracellular cAMP detected in two-day IL-2 or IL-15 activated NK cells before and 5 minutes after the addition of PGE2 (1 µM) (n=3, biological replicates).

E) mRNA levels of selected PGE2 response elements as measured by qPCR in NK cells (n=3-4, biological replicates).

F) Fold change comparing flow cytometry mean fluorescence intensity (MFI) values of PDE4A expression between IL-2 and IL-15 activated NK cells after two days activation with cytokines (n=5, biological replicates).

G) Representative images showing PDE4A expression in NK cells activated by IL-2 or IL-15 for two days. Scale bar: 10 μm.

Data information: Data were analyzed using a paired parametric t test (*, p<0.05, **, p<0.01, ***, p<0.001). Each circle represents an individual experiment. Error bars are depicted as standard deviation.
Phosphodiesterase 4A confers resistance to PGE2-mediated suppression in CD25+/CD54+ NK cells (2020)
Figure 2

Figure 2. Inhibition of mTOR reduces PDE4A expression and sensitizes NK cells to PGE2-mediated suppression

A-B) NK cells were activated with IL-2 or IL-15 for two days in the presence or absence of PGE2 (1µM) and analyzed for A) the levels of phosphorylated S6 (pS235/pS236), STAT3 (S727), STAT5 (Y694), Akt (S473) (n=4, biological replicates) and B)16 categories of phosphorylation array showed. Red box indicates category 1 (pS6+, pSTAT3+, pSTAT5+, pAKT+).

C) Relative changes in frequency of NK cells expressing pS6 in the presence and absence of PGE2 (n=4, biological replicates).

D) Flow cytometry analysis of PDE4A in the presence or absence of Torin-1 (1µM) (left, n=7, biological replicates) with representative histograms (right).

E) NK cell-mediated cytotoxicity of K562 cells by 4-hour chromium-release assay of two-day cytokine activated NK cells in the presence or absence of Torin-1 (1µM) and PGE2 (1µM) (n=6, biological replicates). E:T ratio=5:1.

F, G) (F) Flow cytometry analysis of pS6 and (G) 4-hour chromium-release assay of PGE2-treated NK cells against K562 measured after 48 hours treatment with Roflumilast (PDE4i, 1µM) (n=3, biological replicates) E:T ratio=5:1.

Data information: Data were analyzed using a paired parametric t test (*, p<0.05, **, p<0.01). Each circle represents an individual experiment. Error bars are depicted as standard deviation.
Phosphodiesterase 4A confers resistance to PGE2-mediated suppression in CD25+/CD54+ NK cells (2020)
Figure 3

Figure 3. IL-15 induces phenotypical changes and supports the formation of cell clustering

A) Gene ontology enrichment analysis showing immune system process (GO:0002376) pathways for genes with >1.5 fold-change expression between IL-15 and IL-2. The color of the nodes is determined by the significance of the enriched term assessed by 2-sided hypergeometric test with Bonferroni's step-down method for P value correction.

B) Volcano plot showing genes compared after two-days cytokine activated NK cells. Genes with significant changes (p value<0.05) and >1.5 log2 fold change between IL-2 and IL-15 activated NK cells are labeled in red. Genes belonging to cell surface (GO:0009986) are annotated with gene name.

C) Quantification of CD25+ CD54+ population changes after two days treatment (n=4, biological replicates).

D) Representative flow cytometry graphs for CD25 and CD54 staining in IL-2 and IL-15 NK cells in the presence or absence of PGE2.

E) Quantification of the number of cell clusters. NK cell clusters were defined as cell aggregates occupying an area at least 2000 μm2 and an eccentricity of <0.8). (n=4, biological replicates).

F) Representative phase contrast image of IL-2 and IL-15 activated NK cells in the presence or absence of PGE2. Scale bar 50 µm.

G) Chromium-release cytotoxicity assay by two-day cytokine activated NK cells in the presence or absence of PGE2 (1µM) and anti-CD54 (20 µg/ml) (n=4, biological replicates), E:T ratio=5:1.

Data information: Data were analyzed using a paired parametric t test (*, p<0.05, **, p<0.01, ***, p<0.001). Each circle represents an individual experiment. Error bars are depicted as standard deviation.
Phosphodiesterase 4A confers resistance to PGE2-mediated suppression in CD25+/CD54+ NK cells (2020)
Figure 4

Figure 4. CD25+/CD54+ NK cells display high cytolytic capacity in the presence of PGE2

A) NK cells were stimulated with either IL-2 or IL-15 in the presence or absence of PGE2 for two days and thereafter purified as CD25+/CD54+ and CD25-/CD54- and tested in a 51Cr-release assay against K562. (E: T=5:1, n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54- NK cells for each condition.

B-F) NK cells were stimulated with IL-15 and purified as CD25+/CD54+ and CD25-/CD54- and co-cultures with K562 cells (5:1 ratio) for six hours and analyzed for the expression of B) CD107a, C) IFNγ, D) Perforin, E) TRAIL and F) Granzyme B were by flow cytometry (n=4, biological replicates).

G) Flow cytometry analysis of levels (Left=% frequency, right=fold change of mean fluorescence intensity) of phosphorylated S6 (S235/236) in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates).

H) Flow cytometry analysis of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates).

I) Representative image of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells. Scale bar: 10 µm.

J) NK cells were first stimulated with IL-15 for two days in the absence of PGE2, then purified as CD25+/CD54+ or CD25-/CD54- NK cells and tested in a 51Cr-release cytotoxicity assay against K562 cells at an E:T ratio of 5:1 in the presence (filled bars) or absence (open bars) of PGE2 (1 µM) (n=3, biological replicates).

K) Mitochondria mass from purified CD25+/CD54+ and CD25-/CD54- NK cells measured by flow cytometry (n=5, biological replicates).

L) Representative image of Mitotracker Red FM in CD25+/CD54+ NK cells and CD25-/CD54- NK cells. Scale bar: 20 μm.

Data information: Data were analyzed using a paired parametric t test (*, p<0.05, **, p<0.01， ***, p<0.001). Each circle represents an individual experiment. Error bars are depicted as standard deviation. Box and whiskers plots (K): box extends from 25th to 75th percentile, the center line indicates the median and whiskers indicate min and max values.