EMBO/soda-nel huggingface dataset:
DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron depletion (2022)
Figure 6

Figure 6: Reduced DFP-induced mitolysosomes upon DGAT1 inhibition.
a. Schematic for the mito-QC mitophagy reporter system. mCherry-GFP is targeted to the mitochondrial outer membrane via the specific targeting sequence of FIS1. Cytosolic mitochondrial network appears as yellow, due to red-green fluorescence. Upon mitophagy, mitochondria delivered to endolysosomes are distinguished by mCherry-only puncta, whereas GFP does not fluoresce in the acidic microenvironment.

b. Representative photomicrographs of mitophagy reporter cells (human ARPE-19 cells with stable expression of mito-QC) treated with DFP (1mM) for 7 h followed by treatment with LD inhibitors (5 μM) as indicated on the images (i denotes the presence of inhibitor e.g. DGAT1i = DGAT1-inhibition). Cells were fixed after 24 h of DFP treatment. Mitochondrial networks are visible in yellow and mitolysosomes as red-only puncta signifying mitophagy. Nuclei are counterstained with Hoescht33342. Scale bar = 5 μm.
c. Mitophagy quantification for experiments in 5a. Mitophagy index per cell is measured as a ratio of the number of mitolysosomes per mitochondrial content. Graph generated using the mean of each biological replicate ± SEM (n=7 with at least 70 cells quantified per biological replicate for each condition). Mean area of mitolysosomes per cell and mean number of mitolysosomes per cell for experiments in 5a.

d. Representative flow cytometry assay of mito-QC SH-SY5Y cells treated with DMSO or LD inhibitors (DGAT1/2i, 5 μM) for 24 h, in presence or absence of DFP. A decrease/increase in GFP/mCherry expression is quantified to measure the percent of cells undergoing mitophagy (indicated by number in each panel).

e. Combined quantitation of mito-QC flow data is represented by mean percentage of cells undergoing mitophagy from three independent experiments ± SEM. Statistical analysis was performed with one-way ANOVA and Tukey's multiple comparisons test. ****=P<0.0001; **=P<0.01.

f. Representative confocal photomicrographs showing the pattern of endogenous mitochondrial NIX in human ARPE-19 cells treated with DFP ±DGAT1i/2i. Samples were immunolabelled with primary antibodies to NIX (green) and ATPB (red). Scale bar = 2 μm.

g. Representative confocal photomicrograpghs of Cathepsin-reactive endolysosomes in human ARPE-19 cells treated with with DFP ±DGAT1i/2i. Inset shows examples of lysosomal phenotypes. Scale bar = 5 μm.

h. Colocalization quantitation for experiments shown in 6g. One-way ANOVA with Bonferroni's post hoc test. ****P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates.

i. Colocalization quantitation for ULK1/WIPI2 immunolabelling in human ARPE-19 cells treated with DFP ±DGAT1i/2i. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates.

j. Quantitation of lysosome positioning in DFP ±DGAT1i/2i. One-way ANOVA with Bonferroni's post hoc test. ****=P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates.
DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron depletion (2022)
Figure 7

Figure 7: Phenotypic effects of DGAT1 (mdy) depletion on mitophagy in vivo.
a. Phenotypic assessment of DGAT1/mdy knockdown by d**ifferent RNAi transgenes by GAL4 drivers ubiquitously or in a tissue-specific manner.

b. Schematic for the matrix-QC mitophagy reporter system. mCherry-GFP is targeted to the mitochondrial matrix via the specific COXVIII targeting sequence. As for mito-QC, the cytosolic mitochondrial network appears in yellow, due to red-green fluorescence. Upon mitophagy, mitochondria delivered to endolysosomes are distinguished by mCherry-only puncta, whereas GFP does not fluoresce in the acidic microenvironment.

c. Representative photomicrographs of larval neuronal cell bodies expressing the matrix-QC reporter and RNAi for mdy or control driven with nSyb-GAL4. GFP is shown in green, mCherry is shown in magenta. Mitolysosomes (mCherry-only puncta) are shown in greyscale. Scale bar = 5 μm.

d. Quantitative analysis of mitophagy (mitolysosome per neuronal cell body). Data are shown as violin plot with median (dashed line) and quartile range (dotted lines); n = 5-6 animals, with 23-40 cells per animal. One-way ANOVA with Šidák's post-test correction for multiple samples; **=P<0.01

7e. Analysis of locomotor behaviour (climbing) in animals with neuron-specific (nSyb-GAL4) knockdown of mdy or control. Bars show mean ± 95% CI; n = 40, 57 and 45 animals, respectively. Kruskal-Wallis test with Dunn's post-hoc correction; ****=P<0.0001.
Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and
myopathy (Danon disease) (2000)
figf2

Figure 2: Representation of the mutant LAMP-2b.

‘Normal’ represents the LAMP-2b amino-acid sequence. Because
patient 1, who developed full DD symptoms, has a mutation in exon 9b, the
LAMP-2b isoform is most probably responsible for DD. Residue 265, one of the
putative disulphide-bond-creating cysteines, and two glycosylation sites are
lost by exon-6 skipping in patient 3, which probably results in a significant
change in the secondary structure. Black boxes represent the loops created
between two cysteine residues flanking each of the four loop regions; Y depicts
a glycosylation site; hatched boxes show altered amino acids caused by frameshift
mutations; dashes represent missing amino acids due to exon-6 skipping in
patient 3. All of the other mutant proteins lack the transmembrane domain
(grey bar) because of nonsense or frameshift mutations. Thus, those molecules
cannot function as lysosomal ‘membrane’ proteins.
Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and
myopathy (Danon disease) (2000)
figf3

a, Skeletal muscle. Muscle extracts from patients 1–6 and
10 and a control were blotted and labelled with antibodies against LAMP-2
(top panel) and β-actin (bottom panel). LAMP-2 is deficient in all muscles
examined except the sample from patient 1 that showed a trace amount of protein,
which is probably the LAMP-2a isoform. In support of this idea, LAMP-2b is
the principal LAMP-2 species in skeletal muscle6. Numbers denote
patients as in Table 1; C, control. b, Heart. Protein was extracted
from cardiac muscle of patient 4 and a control, blotted onto a nitrocellulose
membrane, and labelled with the antibodies against LAMP-2 (too panel) and β-actin
(bottom panel). LAMP-2 is deficient also in cardiac muscle. C, control; 4,
patient 4.
Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and
myopathy (Danon disease) (2000)
figf4

Serial transverse sections of the biopsied muscle from six DD patients,
and samples from a XMEA patient and a normal control, were immunostained with
antibodies against LAMP-2 and limp-I. LAMP-2 is absent in all DD muscles (patient
6 shown here), whereas it is present in the XMEA muscle. The antibody against
limp-I highlighted vacuoles in both DD and XMEA muscles. LAMP-1 antibody faintly
stained both DD and XMEA muscles, suggesting that the expression level of
LAMP-1 might be lower in skeletal muscle than LAMP-2 or limp-I (data not shown).
The muscle from the patient without a lamp-2 mutation also showed LAMP-2
deficiency on immunohistochemistry similar to genetically proven DD patients
with primary LAMP-2 deficiency (data not shown). The immunohistochemical stains
of LAMP-2, limp-I, and LAMP-1 in normal muscle shows numerous tiny particles
and accentuates type 1 fibres. H&E, haematoxylin and eosin, original magnification
×150.
Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor (2019)
Figure 1

Figure 1. AGO1 transcript undergoes translational readthrough.

(A) Alignment of amino acid sequences potentially encoded by the proximal 3′UTR of AGO1 mRNA. Conserved residues are shown in grey background; sequence of the peptide used to raise an antibody against the putative readthrough product (Ago1x) is shown. *, position of in-frame stop codons.

(B) Schematic of the construct used in luciferase-based readthrough assays. Last 696 nucleotides of coding sequence of AGO1 and 99 nucleotides of the ISR were cloned upstream and in-frame with firefly luciferase (FLuc).

(C) Demonstration of translational readthrough of AGO1 using luciferase-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co-transfected renilla luciferase (RLuc). FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0001.

(D) Significance of the identity of the canonical stop codon in AGO1 translational readthrough. AGO1-ISR-FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above. FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0002; **, P = 0.0004.

(E) First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough. AGO1-TGA-FLuc constructs with different lengths of ISR (all in-frame with AGO1 and FLuc) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate. FLuc activity reflects readthrough activity. **, P = 0.012; *, P = 0.017.

(F) Demonstration of translational readthrough of AGO1 using fluorescence-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence. Scale bar, 50 um.

(G) The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs. Fluorescence intensity was measured by flowcytometry. ***, P = 0.007.

Data information: Bar graphs (mean ± S.E.) are representatives of at least three independent experiments done in triplicates. Statistical significance was calculated using Student's t test. ISR, Inter-stop codon region; ∆ISR, construct without ISR.
Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor (2019)
Figure 2

Figure 2. Detection of endogenous readthrough product of AGO1 (Ago1x)

(A) Schematic showing the difference between two isoforms of AGO1.

(B) Detection of endogenous Ago1x by Western blot in HEK293 cells using anti-Ago1x antibody and pre-immune serum.

(C) Western blot of lysates from HEK293 and HeLa cells using anti-Ago1x antibody pre-treated with Ago1x-specific peptide.

(D) Expression of Ago1x in HEK293 cells transfected with three AGO1-specific siRNAs (1,2,3). qRT-PCR analysis of AGO1 transcript expression in these cells is shown below.

***, P < 0.0001 (Student's t test). Bars: mean ± S.E; n=3;

(E) Proportion of Ago1x isoform in HEK293 cells. To separate Ago1x and Ago1 (~ 3 kDa difference), the cell lysate was electrophoresed for 3.5 h in 10% polyacrylamide gel. Proportion of Ago1x and canonical isoform (Ago1) was calculated by densitometric analysis. Results from three independent experiments are shown as a bar graph (mean ± S.E.).

(F) Expression of Ago1x in mouse organs.

(G) Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2-OS cells. Ribosome profiling data was taken from NCBI's sequence read archive (SRA Accession no. SRR1257257) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section. Zoomed area near the inter-stop codon region is shown in the inset. Position of canonical stop codon and the downstream in-frame stop codon are shown. Detection of Ago1x by Western blot in U2-OS cells is shown (right).
Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor (2019)
Figure 3

Figure 3. Translational readthrough of AGO1 is regulated by let-7a miRNA.

(A) Sequence alignment showing the binding site of let-7a miRNA in the inter-stop codon region of AGO1.

(B) Effect of let-7a inhibitor on translational readthrough of AGO1. let-7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.01.

(C) Effect of let-7a overexpression on translational readthrough of AGO1. HeLa cells were stably transfected with pri-let-7a overexpression construct. Ago1x expression was determined by Western blot. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.007.

(D) let-7a inhibitor reduces the AGO1 readthrough efficiency. HeLa cells were transfected with let-7a inhibitor or control inhibitor along with AGO1-TGA (or GCA)-ISR-FLuc construct. Readthrough was quantified by measuring FLuc activity, which was normalized to co-transfected renilla luciferase activity. FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown (Right). *, P = 0.027.

(E) AGO1 ISR with mutated let-7a binding site shows reduced translational readthrough activity. Assay was done as described above in HeLa cells. Mutated sequence is shown (top). ***, P = 0.008.

(F) AGO1 ISR with displaced let-7a binding site (AGO1-TGA-disISR-FLuc) shows reduced translational readthrough activity. let-7a binding site was moved 18 nucleotides downstream of the stop codon. Readthrough assay was done as described above in HeLa cells. ***, P < 0.0001.

Data information: Bar graphs (mean ± S.E.) are representatives of at least three independent experiments. Statistical significance was calculated using paired Student's t test.
Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor (2019)
Figure 4

Figure 4: Translational readthrough in heterologous context

(A) AGO1 ISR can drive translational readthrough in a heterologous context of RHOA. AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RHOA coding sequence (582 nucleotides) and the readthrough assay was done as described above in HeLa cells. 3′UTRRho, 159 nucleotides in the proximal 3′UTR of RHOA (NM_001664); ISRAgo1, Inter-stop codon region of AGO1. ***, P < 0.0001.

(B) AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase (RLuc). AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells (left) and in vitro using rabbit reticulocyte lysate system (right). **, P = 0.006; ***, P = 0.0001.

(C) Tethering of N-peptide-tagged Ago1 or Ago1x can induce readthrough of AGO1. Two BoxB elements were cloned downstream of the AGO1 and upstream of the in-frame FLuc as shown in the schematic. This construct was transfected in HeLa cells expressing N-peptide-tagged or FLAG-tagged Ago proteins as indicated. Readthrough was measured as described above. *, P = 0.035 (with Welch correction); **, P = 0.016.

Data information: Bar graphs (mean ± S.E.) are representatives of at least three independent experiments done in triplicates. Statistical significance was calculated using Student's t test.
Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor (2019)
Figure 5

Figure 5: Ago1x can load miRNAs onto target mRNA

(A) Linear regression analysis of miRNAs co-immunoprecipiated with Ago1 and those co-immunoprecipiated with Ago1x in HeLa cells. Reads per million (RPM) values of 395 miRNAs, which were common to Ago1 and Ago1x are shown.

(B) qRT-PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells. Bars show mean of three experiments ± S.E.

(C) RT-PCR results showing PTEN and MYC mRNAs co-immunoprecipitated with Ago1 or Ago1x in HeLa cells.

(D) Result of co-immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells.
Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor (2019)
Figure 6

Figure 6: Ago1x does not interact with GW182

(A) Result of co-immunoprecipitation experiment in HeLa cells shows that GW182 is co-immunoprecipitated with Ago1, but not with Ago1x.

(B) Confocal immunofluoresence images of HeLa cells show co-localization of GW182 with Ago1, but not with Ago1x. White circles indicate zoomed areas.

(C) Confocal microscopy images of proximity ligation assay (PLA) in HeLa cells showing interaction of GW182 with Ago1, but not with Ago1x. Rabbit anti-GW182 and Mouse anti-FLAG antibodies were used to achieve proximity ligation. Interaction is indicated by red dots that results from proximity ligation. Quantification of dots per cell is shown (right). Each black circle represents a microscopic field. Mean ± S.E. are shown as horizontal lines (n=10 fields for vector control; n=12 fields for Ago1; n=11 fields for Ago1x). At least 100 cells were counted for dots in each category. ***, P < 0.0001 (Mann-Whitney test).

All results are representative of three independent experiments.
Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor (2019)
Figure 7

Figure 7: Ago1x does not inhibit translation of target mRNAs

(A) Schematic showing the principle of N-peptide and BoxB element-based assay to study translation repression ability of Ago proteins. N-peptide-tagged Ago1 and Ago2 proteins bind the BoxB elements in the 3′UTR of renilla luciferase (RLuc) and bring down their translation.

(B) Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Bar graph (mean ± S.E.) is a representative of four independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). *, P = 0.018; #, P = 0.041; $, P = 0.013. Numbers on the bars represent % change in relative luciferase activity.

(C) Expression of RLuc protein in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). RT-PCR analysis for RLuc mRNA level is shown (bottom). **, P = 0.001.

(D) Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged Ago2 (N-HA-Ago2) or N-HA-tagged chimeric Ago2 appended with the ISR region of Ago1x (N-HA-Ago2-ISRAgo1) as indicated. Bar graph (mean ± S.E.) is a representative of three independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). ***, P < 0.0001; **, P = 0.0004.

Data information: Statistical significance in all bar graphs was calculated using Student's t test.
Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor (2019)
Figure 8

Figure 8: Overexpression of Ago1x increases global translation

(A) Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of four independent experiments is shown as bar graph (mean ± S.E.). Puromycin densities were normalized to ponceau S stain densities. *, P < 0.029 (Mann-Whitney test).

(B) [35S]-methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). Autoradiogram densities were normalized to Coomassie stain densities. *, P < 0.029 (Mann-Whitney test).
Calcium flux control by Pacs1-Wdr37 promotes lymphocyte quiescence and lymphoproliferative diseases (2021)
Figure 1

FIGURE 1: Pacs1 is required for normal numbers of circulating lymphocytes

(A) Super-pedigree mapping of two mutations in Pacs1 that were linked to peripheral B cell deficiency. Insert shows peripheral B cell deficiency in the endive (N=22 REF, 28 HET, 10 VAR) and chicory (N=24 REF, 15 HET, 3 VAR) pedigrees. Horizontal bars indicate mean values. Ordinary one-way ANOVA with Tukey's multiple comparisons test. Ns = not significant, **P < 0.01, and ***P < 0.001. Protein domain model shows the encoded location of the ENU alleles.

(B) A 1 bp insertion in Pacs1 using CRISPR/Cas9 leads to loss of Pacs1 protein (red arrowhead).

(C) Peripheral blood immune cell counts from Pacs1+/+ and Pacs1−/− mice. Horizontal bars indicate mean values. Two-tailed unpaired t test, *P < 0.05, **P < 0.01, and ***P < 0.001.

(D) Absolute numbers of lymphocytes subpopulations in the bone marrow, thymus, and spleen. B cell development in the bone marrow was assessed by FACS analysis for surface expression of: B220+CD43+CD19−IgM−IgD− (pre-pro B); B220+CD43+CD19+IgM−IgD− (pro B); B220+CD43−CD19+IgM−IgD− (pre B); CD19+IgM+IgD− (immature); CD19+IgM+IgD+ (mature). T cell development in the thymus was assessed by FACS analysis for surface expression of: CD4−CD8− (double negative, DN); CD4+CD8+ (double positive, DP); CD4+CD8− (CD4 single positive, SP); CD4−CD8+ (CD8 SP). Splenic B cell populations were assessed by FACS analysis for surface expression of: B220+CD21+CD23+ (follicular B cells, FOB); B220+CD21+CD23low (marginal zone B cells, MZB). Each symbol represents an individual mouse. Horizontal bars indicate mean values. Two-tailed Mann-Whitney test, ns = not significant, *P < 0.05, and **P < 0.01.

(E) Proportion of cell populations derived from Pacs1+/+;CD45.1 and Pacs1−/−;CD45.2 donors during competitive bone marrow reconstitution. Populations were determined based on the same markers as in (C) with the added congenic markers CD45.1 and CD45.2. Each symbol represents an individual recipient. Results are representative of two independent transplant experiments. Two-tailed unpaired t test, ns = not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.

(F) Measurement of cell death with Annexin V staining in FOB and MZB cells from the spleens of Pacs1+/+ and Pacs1−/− mice. Results are representative of two independent experiments. Horizontal bars indicate mean values. Two-tailed unpaired t test, ns = not significant, **P < 0.01.
Calcium flux control by Pacs1-Wdr37 promotes lymphocyte quiescence and lymphoproliferative diseases (2021)
Figure 3

FIGURE 3: Wdr37 forms a mutually stabilizing complex with Pacs1

(A) Super-pedigree mapping of two mutations in Wdr37 that are linked to peripheral B cell deficiency. Insert shows peripheral B cell deficiency in radical (N=12 REF, 18 HET, 4 VAR) and profound (N=14 REF, 15 HET, 6 VAR) pedigrees. Horizontal bars indicate mean values. Ordinary one-way ANOVA with Tukey's multiple comparisons test, ns = not significant, ***P < 0.001.

(B) Co-immunoprecipitation of HA-tagged Pacs1 by FLAG-Wdr37 and HA-Wdr37 by FLAG-Pacs1 in co-transfected 293T cells.

(C) Western blot for Pacs1 and Wdr37 expression in peripheral blood cells from WT­, Pacs1−/−, and Wdr37−/−­ mice.

(D) B and T cell peripheral blood counts in Wdr37−/− mice. Horizontal bars indicate mean values. Two-tailed unpaired t test, ***P < 0.001.

(E) Wdr37+/+ and Wdr37−/− splenocytes were labeled with Indo-1, stained for cell surface markers to identify FOB cells, and stimulated with the indicated amounts of anti-IgM (black arrowhead). Normalized traces from three (2.5 mcg/ml anti-IgM) or four independent experiments (10 mcg/ml and 5 mcg/ml anti-IgM) are shown (Wdr37+/+ gray, Wdr37−/− pink). Mean Ca2+ flux for each genotype is overlaid in bold (Wdr37+/+ black, Wdr37−/− red).

(F) Maximum Ca2+ flux at each anti-IgM concentration is shown. Two-tailed paired t test, *P < 0.05, **P < 0.01.

(G) Wdr37+/+ and Wdr37−/− FOB cells were labeled with Indo-1 and stimulated in Ca2+ free buffer with 5 mcg/ml anti-IgM (black arrowhead) followed by addition of 2 mM Ca2+. Normalized traces from four independent experiments are shown with mean Ca2+ flux overlaid in bold.

(H) Maximum Ca2+ flux after stimulation under Ca2+ free conditions and after Ca2+ was added back. Two-tailed paired t test, ns = not significant, *P < 0.05.