EMBO/soda-nel huggingface dataset:
Synergistic Recruitment of UbcH7~Ub and Phosphorylated Ubl Domain Triggers Parkin Activation (2018)
Figure 5

Figure 5. The pUbl domain and UbcH7-Ub synergistically modulate catalytic C431 reactivity

Exposure of the catalytic Cys431 is parkin as assessed by reaction with a UbVS probe. The different stages of activation using parkin, parkin:pUb, pParkin:pUb and pParkin:pUb in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-10 minutes) and visualized by SDS-PAGE.

Relative percentages of pParkin and the pParkin-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of pParkin-Ub, parkin and UbVS/pUb bands. Error bars represent standard deviation from the average for duplicate measurements.

Exposure of the catalytic Cys431 is R0RBR parkin as assessed by reaction with a UbVS probe. The different stages of activation using R0RBR, R0RBR:pUb, R0RBR:pUb:pUbl and R0RBR:pUb:pUbl in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-60 minutes) and visualized by SDS-PAGE

Relative percentages of R0RBR and the R0RBR-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of the R0RBR-Ub, R0RBR and UbVS/pUb/pUbl bands.
TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells (2021)
Figure 1

Figure 1. Quantification of SARS-CoV-2 infection.

(A, B) Cells were lysed and analyzed by SDS-PAGE and western blotting under nonreducing conditions (A) or reducing conditions (B). TMPRSS2 levels are expressed as percentages of TMPRSS levels in Calu-3 cells normalized to levels of EF2. *1 indicates TMPRSS2 (A) and pro-cathepsin L (B), and *2 shows cleaved TMPRSS2 (A) and cathepsin L (B). A549*, ACE2-expressing A549 cells; EF2, elongation factor 2.

(C) Vero and Caco-2 cells were infected with SARS-CoV-2 at MOIs of 0.2 for 8 h. Infected cells were then permeabilized and immunostained for the intracellular SARS-CoV-2 nucleoprotein (NP, red). Nuclei were stained with Hoechst (blue) before imaging by fluorescence confocal microscopy. Scale bars: 100 µm.

(D) Vero and Caco-2 cells were exposed to SARS-CoV-2 at MOIs of 0.3 and 0.2, respectively, and harvested 16 h later. After fixation and permeabilization, infected cells were stained with the primary mAb against NP. Infection was analyzed by flow cytometry. SSC-A = side scatter-area.

(E) Infection of Vero and Caco-2 cells was monitored over 24 h using the same flow cytometry-based assay used for the experiment shown in panel D. Infection is given as the total fluorescence associated with the NP protein-positive cells. MFI, mean fluorescence intensity. n = 3.

(F) SARS-CoV-2 mRNA levels were quantified by qRT-PCR in both Vero and Caco-2 cells infected at MOIs of 0.3 and 0.4, respectively, for up to 24 h. n = 3.

(G) Supernatants from infected cells were collected during the time course in F and assessed for the production of new infectious viral particles using a TCID50 assay on naïve Vero cells. n = 3-6.

Data information: images are representative of at least three independent experiments. (E, F) Results are representative of 3 independent experiments and expressed as mean ± standard error of mean (SEM) of 3 biological replicates.
TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells (2021)
Figure 2

Figure 2. SARS-CoV-2 makes differential use of host cell proteases for infectious penetration.

(A, B) Cells were pretreated with the indicated concentrations of aprotinin (A) and SB412515 (B), which are inhibitors of TMPRSS2 and cathepsin L, respectively. Infection of Calu-3, Caco-2, A549*, and Vero cells with SARS-CoV-2 at MOIs of 0.3, 0.4, 0.2, and 0.3, respectively, was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 2-4 biological replicates.

(C, D) SARS-CoV-2 particles were bound to A549* and Vero cells (MOIs 0.2 and 0.3, respectively) (C) or Calu-3 and Caco-2 cells (0.6 and 0.5, respectively) (D) on ice for 90 min and subsequently warmed rapidly to 37°C to allow infectious penetration. SB412515 (10 µM, C) or aprotinin (30 µM, D) was added at different times post warming to block further proteolytic activation. Infection was analyzed by flow cytometry, and data were normalized to samples where protease inhibitors had been omitted. n = 2.

Data information: (A, B) data are expressed as mean ± SEM from 2 independent experiments. (C, D) Results are representative of 2-3 independent experiments and expressed as mean ± SEM of 2 biological replicates.
TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells (2021)
Figure 4

Figure 4. TMPRSS2 drives pH- and cathepsin L-independent SARS-CoV-2 entry.

(A) TMPRSS2-overexpressing and parental A549* cells were lysed and subjected to SDS-PAGE and western blot analysis under reducing conditions. *1 and *2 indicate the full-length and cleaved forms of TMPRSS2, respectively.

(B-D) A549* cells expressing or lacking TMPRSS2 were pretreated with the indicated concentrations of camostat mesylate (B), SB412515 (C), and bafilomycin A1 (D). Infection with SARS-CoV-2 (MOI ~0.2) was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 5-6 biological replicates.

Data information: images are representative of at least 3 independent experiments. Data are all expressed as mean ± SEM from 3 independent experiments.
TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells (2021)
Figure 5

Figure 5. SARS-CoV-2 relies on late endosomal maturation for infection.

(A) EGFP-Rab7a wild-type (wt), Q79L (constitutively active mutant), and T22N (dominant-negative mutant) were transiently expressed in Vero cells. The cells were then infected with SARS-CoV-2 at an MOI of ~0.3. Using flow cytometry, cell populations with levels of EGFP-Rab7a expression varying by roughly one-log increments were selected, and the proportion of infected cells within each population was quantified at 8 hpi. Data were normalized to that of the cell population with the lowest EGFP-Rab7a intensity. Unpaired t-test with Welch's correction was applied. *, p < 0.05; **, p < 0.01. n = 5-6 biological replicates.

(B, C) Vero and A549* cells were pretreated with colcemid (B) or MG-132 (C) at the indicated concentrations and subsequently infected with SARS-CoV-2 (MOIs ~0.3 and 0.2, respectively) in the continuous presence of inhibitors. Infection was analyzed by flow cytometry, and data were normalized to samples where inhibitors had been omitted. n = 3-4 biological replicates. Unpaired t-test with Welch's correction was applied. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

(D) SARS-CoV-2 particles were bound to A549* and Vero cells (MOIs of 0.2 and 0.3, respectively) on ice for 90 min and then switched rapidly to 37°C to allow infectious penetration. MG-132 (3.7 µM) was added to cells at the indicated times to block further late endosomal maturation. Infection was analyzed by flow cytometry, and data were normalized to that of control samples that had not been treated with MG-132. n = 2.

(E) As in panel B but using a MOI of 0.4 and Caco-2 cells instead of Vero cells. n = 6 biological replicates.

(F) As in panel C but using a MOI of 0.3 and 0.4 and Calu-3 and Caco-2 cells. n = 2-8 biological replicates.

(G) The timing of the MG-132-sensitive step during SARS-CoV-2 infectious entry into Calu-3 and Caco-2 cells was assayed as detailed in D but using 60 µM MG-132 for Caco-2 cells and MOIs of 0.5 and 0.6, respectively. n = 2.

(H) A549* cells expressing or lacking TMPRSS2 were pretreated with MG-132 at the indicated concentrations and subsequently infected with SARS-CoV-2 (MOI ~0.2) in the continuous presence of the drug. The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to that from control samples for which MG-132 had been omitted. n = 6 biological replicates.

Data information: (A to C, E, F, and H) data are expressed as mean ± SEM from 2-4 independent experiments. (D and G) Results are representative of at least 2 independent experiments and expressed as mean ± SEM of 2 biological replicates.
TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells (2021)
Figure 6

Figure 6. Proteolytic processing triggers SARS-CoV-2 membrane fusion.

(A) SDS-PAGE and western blot analysis of SARS-CoV-2 particles treated with trypsin or furin under reducing conditions. S was detected using a rabbit antibody against the SARS-CoV-2 S2 region.

(B) SARS-CoV-2 regions S0 (uncleaved) and S2 (cleaved) were semiquantified from (A), and the proportion of S2 on viral particles is shown as (ρ) = S2/S0+S2. n = 3.

(C) SARS-CoV-2 was subjected to pretreatment with trypsin and furin for 15 min at 37°C prior to infection of Caco-2 and Vero cells (MOIs of 0.4 and 0.3, respectively). Infected cells were quantified by flow cytometry as described in Fig. 1D. Data were normalized to samples not pretreated with trypsin. n = 4-6 biological replicates.

(D) Depicts the cell-cell fusion model employed in this study. (1) or (2) indicate treatment with either exogenous proteases or low pH buffers; CMFDA, cytosolic green dye; IF anti-NP, immunofluorescence staining against the SARS-CoV-2 nucleoprotein.

(E) A549* expressing or lacking TMPRSS2 cells and Vero cells were first infected with SARS-CoV-2 at MOIs of 0.1 and 0.2, respectively, for 24 h and then cocultured for 5 h along with target cells, not infected but prestained with CMFDA, a cytosolic green dye. Cells were subsequently treated with trypsin or furin for 5 min at 37°C and left to coculture for an additional hour at 37°C. After fixation, nuclei were stained with Hoechst (blue), and infected cells were subjected to immunofluorescence staining against SARS-CoV-2 nucleoprotein (magenta). Samples were ultimately imaged by confocal fluorescence microscopy. White stars indicate syncytia with at least four nuclei. Scale bars: 100 µm.

(F) Images of microscope fields obtained in (E) were quantified [A549*: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 80, and n (furin) = 60; A549* TMPRSS2: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 79, and n (furin) = 60; Vero: n (no virus) = 40, n (no protease) = 112, n (trypsin) = 114, and n (furin) = 60]. The fusion index is given as f = 1 - [(number of cells in a field after fusion)/(number of nuclei)].

Data information: Images are representative of three independent experiments. (B) Data are expressed as mean ± SEM from 3 independent experiments. (C) Data are expressed as mean ± SEM from 2-3 independent experiments. (F) Results are expressed as mean ± SEM of 3 independent experiments.
TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells (2021)
Figure 8

Figure 8. SARS-CoV-2 no longer requires endosomal acidification after proteolytic processing.

(A) Trypsin- or thermolysin-activated SARS-CoV-2 was allowed to infect A549* and Vero cells at MOIs of 0.2 and 0.3, respectively, in the continuous presence of bafilomycin A1. Infection was quantified by flow cytometry, and data were normalized to that from control samples not exposed to the inhibitor. n = 2-4 biological replicates.

(B) Binding of trypsin-activated SARS-CoV-2 to A549* and Vero cells (MOI ~0.2 and 0.3, respectively) was synchronized on ice for 90 min. Subsequently, the cells were rapidly shifted to 37°C to allow penetration. NH4Cl (50 mM) was added at the indicated time to neutralize endosomal pH and block the acid-dependent step of SARS-CoV-2 infectious penetration. Infected cells were analyzed by flow cytometry, and data were normalized to samples where NH4Cl had been omitted. n = 2.

(C) SARS-CoV-2 was first treated with trypsin or thermolysin and then allowed to infect A549* and Vero cells (MOIs ~0.2 and 0.3) in the continuous presence of SB412515 (cathepsin L inhibitor). Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where SB412515 had been omitted. n = 4 biological replicates.

Data information: (A and C) data are expressed as mean ± SEM from 2 independent experiments. (B) Results are representative of 2 independent experiments and expressed as mean ± SEM of 2 biological replicates.
Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy (2021)
Figure 2

Figure 2. PRKAG2 S124 is directly phosphorylated by ULK and AMPK and regulated by serum status.

A) Immunopurified C-terminally GFP tagged PRKAG2 N-terminal tail fragments (amino acids 1-136, 1-242) with deletion of Δ122-124 or without (WT) were incubated with full length Ulk1 wild type (WT) or kinase inactive (KI) for in vitro kinase assays. Autoradiograms and Coomassie gels reveal 32P incorporation and protein level respectively.

B) GFP-tagged PRKAG2 N-terminal tail fragments (amino acids 1-242) were immunopurified and phosphorylated in vitro by Ulk1 WT or KI. Immunoblot analysis revealed that S124 is phosphorylated by Ulk1 in vitro.

C) Full length FLAG-PRKAG2 WT or Δ124 were coexpressed with full length Ulk1 WT and incubated in the presence of 1μM MRT68921. Western blot analysis revealed that Ulk1 overexpression strongly promotes S124 phosphorylation in cells.

D) Cells expressing empty vector (EV), or PRKAA1-myc, PRKAB2 and PRKAG2-FLAG (AMPK) alone or with myc-Ulk1 WT (ULK) were cultured in full medium (F), EBSS (St), or serum-free medium (SFM) alone or in the presence of 991 (1µM) or MRT68921 (1μM) for 90 minutes as indicated. 2% Loading control and pull down samples were analysed by Western blot.

Data information: (B, C) Identical samples were analysed on separate blots, separated by dashed lines.
Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy (2021)
Figure 3

Figure 3. VPS15 contains 6 putative ULK substrate residues.

A) VPS15-ZZ was co-expressed with Ulk1 and Ulk2 wild type (WT) or kinase inactive (KI) before analysis by Western blot. An electrophoretic mobility shift in VPS15-ZZ was noted with WT only.

B) VPS15 is depicted schematically, with the N-terminal myristate and pseudokinase, HEAT and WD40 domains. The 6 potential ULK substrates are annotated below in red.

C) Alignment of human VPS15 sequences spanning each of the putative ULK substrates. Simplified rendering of the ULK consensus motif stipulating hydrophobics at positions -3, +1 and +2 is shown above (Φ = hydrophobic). These positions are highlighted in the sequence alignment (red -3, blue +1/+2) with the phosphoacceptor in green.

D) Human VPS34 complex II homology model showing VPS15 (green), VPS34 (grey), Beclin1 (beige) and UVRAG (pink) and potential ULK substrates (red spheres). (Left) Y-shaped VPS34 complex II in standard orientation with N and C termini of VPS15 labelled. The blue dashed line represents a lipid membrane, positioned close to the membrane binding (left- and right-hand tips of Y) and lipid kinase (right hand tip of Y) domains of the complex. (Right) Rotated 90 degrees on horizontal axis towards point of view.
Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy (2021)
Figure 4

Figure 4. VPS15 phosphorylation affects autophagy initiation and PI3P formation

A) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot. LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA-derived clones.

B) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr, fixed and imaged for WIPI2 (green). Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles (red asterisks). Quantification of WIPI2 spots, mean +/- SEM, n=3.

C) GFP-2xFYVE was overexpressed in control (A3) and VPS15 KO clones. The GFP-2xFYVE puncta were abolished in VPS15 KO clones.

D) Wild type (WT), 6SA or 6SE VPS15-HA, or empty vector (EV), were expressed in VPS15 KO or HEK293A cells. Cells were starved for 1 hr and the distributions of WIPI2 (green), HA (red) and LC3 (blue) were assessed, mean +/- SEM, n=4.

E) (Top) VPS34 CI incorporating VPS15 WT (black), 6SA (blue) or 6SE (pink) were purified and thermal stability was assessed. The first derivative of the ratio of tryptophan emission at 330 and 350nm (Y axis) is plotted against temperature (X axis, °C) with threshold melting temperatures annotated. (Bottom) Representative Coomassie stained gel shows relative protein levels used.

F) In vitro lipid kinase activities of WT-, 6SA- or 6SE-reconstituted VPS34 complex I on GUVs (giant unilamellar vesicles) were assessed. Representative images show GUV membranes (Lissamine Rhodamine-PE, green) and PI3P (AlexaFluor647-PX, red). Bottom left: VPS34 complex activities over time measured by recruitment of AlexaFluor647-PX to GUVs are shown. Bottom right: kinase reaction rates for GUVs phosphorylated with WT, 6SA or 6SE complexes, taken from linear portion of reactions. Representative data from 1 of 4 independent repeats are shown, with mean +/-SD. In this experiment, 67 GUVs were analysed in total (15 from WT, 17 from 6SA and 35 from 6SE).

Data information: (B, D) ** p<0.01, ***p<0.001 [one-tailed ANOVA]. (F) ***p<0.001, n.s. not significant, [student's T test]. Scale bars 10µm (B, D) and 5µm (C, F).
Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy (2021)
Figure 5

Figure 5. Serine 861 is the major ULK phosphoacceptor in VPS15.

A) VPS34 CI was phosphorylated in vitro by Ulk1 1-427. Coexpressed VPS34, VPS15, Beclin1 and ATG14-ZZ were immunopurified and incubated with Ulk1 1-427 in vitro. The samples were then separated by SDS PAGE and transferred onto PVDF membrane. In vitro phosphorylated proteins were visualised by autoradiography before VPS15 was visualised by immunoblotting, revealing that VPS15 and ATG14-ZZ were phosphorylated in this assay.

B) All subunits for VPS34 CI containing ATG14-ZZ or CII containing UVRAG-ZZ were phosphorylated in vitro by WT or KI Ulk1 1-427 as indicated in A. Autoradiogram shows VPS15, ATG14-ZZ, UVRAG-ZZ and Ulk1 1-427 are phosphorylated. Phosphorylation occurred in reaction mixtures containing Ulk1 1-427 WT. The Coomassie-stained gel shows VPS15 levels in assay.

C) WT VPS15 or VPS15 with 6SA, 2SA or individual phosphoacceptors mutated to alanine were incubated in vitro with Ulk1 1-427 and analysed as in B. The Coomassie-stained gel shows VPS15 levels in assay. Mean +/-SEM, n=3.

D) VPS34 CI components expressed with Ulk1 1-427 WT or KI as indicated. After starvation for 1 hr in the presence or absence of MRT68921 (ULK inhibitor), cells were lysed and VPS34 CI coimmunoprecipitated via ATG14-ZZ. VPS15 S861 phosphorylation was increased in the presence of active Ulk1. VPS15 pS861/total VPS15 was quantified, mean +/- SEM, n=3.

E) HEK293A were transfected with VPS34 CII components and Ulk1 1-427 WT or KI and treated as indicated before VPS34 CII co-immunopurification via UVRAG-ZZ. Samples were analysed by Western blot as in D, revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII. Quantifications show mean of 4 independent experiments +/-SEM, ***p<0.001 [one-tailed ANOVA].

F) Representative loading control samples from experiments in D and E were analysed by immunoblot. UVRAG and ATG14, each visualised using the affinity of the ZZ tag for 2° antibodies alone, underwent electrophoretic mobility shifts in the presence of active Ulk1 only, indicative of direct phosphorylation in cells.

Data information: (A, B) dashed lines indicate where autoradiograms were cropped for presentation. * p<0.05., ** p<0.01, *** p<0.001, n.s. not significant, [one-tailed ANOVA].
Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy (2021)
Figure 6

Figure 6. ULK-dependent phosphorylation of serine 861-phosphoryaltion regulates autophagy.

A) HEK293A, VPS15 KO control and stably rescued VPS15 KOs (sgA-B3) were starved for 1 hr before WIPI2 (green), LC3 (red) and DNA (Hoechst, blue) were visualised. Quantification of WIPI2 puncta number per cell, mean +/-SEM, n=5.

B) Stably rescued VPS15 KO (sgA-B3), HEK293A and VPS15 KO control cells were starved for 1 hr with (SB) or without (St) 100nM Bafilomycin A1, or cultured in full media (F). Quantification shows LC3II/Actin, mean +/-SEM, n=5.

C) Autophagic flux examined over a four-hour starvation time-course. LC3II/Actin was quantified, mean +/-SEM, n=6.

Data information: * p<0.05, ** p<0.01, ***p<0.001, [one-tailed ANOVA]. Scale bar 10µm.
Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy (2021)
Figure 7

Figure 7. VPS15 KOs accumulate phosphorylated ULK substrates and aberrant autophagic structures.

A) HEK293A were treated with VPS34-IN1 (1μM) alone or in the presence of MRT68921 (1μM) for the indicated time. Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot. Identical samples were loaded in parallel for VPS34, ATG13, PRKAB2 analysis, indicated by dashed line.

B) HEK293A and VPS15 KOs were fixed and the labelled for p62 (green) and FIP200 (red).

C) HEK293A and VPS15 KOs were fixed and the labelled for ubiquitin (green), Ferritin (red) and p62 (blue).

D) HEK293A and VPS15 KOs were stained for p62 (green), ATG9 (red) and LAMP1 (blue).

E) HEK293A and VPS15 KO cells were fed, or starved for 2 hrs before fixation, and labelling with ULK1 (green), ATG9A (red) and GM130 (blue). Dashed boxes show magnified regions of interest.

Data information: Scale bars 10µm.
NPM1 ablation induces HSC aging and inflammation to develop MDS exacerbated by p53 loss (2022)
Figure 1

Figure 1. Npm1 conditional mutants in the hematopoietic system.

(A) NPM1 expression in CD34+Lin- cells from myelodysplastic syndrome (MDS) patients compared to healthy volunteers (n = 29). Data are shown as mean ± SD. Unpaired two-tailed t-test, *** p<0.001.

(B) Representative staining of NPM1 protein levels in bone marrow samples of MDS or acute myeloid leukemia (AML) with multi-lineage dysplasia (MLD). Insets show representative staining in blasts [B] and in differentiated myeloid cells [e.g., neutrophil, M] with a high magnification. Scale bar, 50 μm.

(C) Percentages of patient samples with NPM1-low and NPM1-normal in healthy, MDS and MLD in AML groups.

(D) Schematic strategy of Npm1 conditional knockout. Excision of exons 1-6 from the Npm1 allele by Cre-recombinase produces an Npm1-knockout allele. Hypomorphic allele (top), NEO-free allele (middle, Flox) and excised allele are shown. The genomic sequence is depicted as a black line, with black boxes representing exons 1-11. Grey box, red triangles and arrows represent an Frt (blue)-flanked neomycin resistance cassette (NEO), the loxP site, and genotyping primers, respectively.

(E) Schema of mating strategy and numbers of viable pups at weaning bearing the 6 possible genotypes for Vav1-Cre. A Chi-square test determined any difference between observed proportions and expected proportions.

(F) Schematic representation of Vec and Vav1 expression during embryogenesis.

(G) Schema of mating strategy and numbers of viable pups at weaning bearing the 6 possible genotypes for Vec-Cre. A Chi-square test determined any difference between observed proportions and expected proportions.