EMBO/sd-character-level-ner huggingface dataset:
acetylation on SUMO chain architecture under heat stress (a) HeLa cells were depleted for endogenous SUMO2/3 by siRNA and transfected with SUMO2T90K (WT or K11Q).Figure 4(a) HeLa cells were depleted for endogenous SUMO2/3 by siRNA and transfected with SUMO2T90K (WT or K11Q). Prior to lysis, cells were heat shocked at 43°C for 1 h. The lysate was digested with LysC, followed by GlyGly-enrichment. Subsequently, samples were measured by LC-MS/MS and analyzed with MaxQuant software. (b) Immunoblotting of samples with α-RGS-His (SUMO variants) and α-PCNA as loading control. (c) Bar graph, comparing the identified SUMO-SUMO2 linkages of control and heat shock samples for SUMO2WT (gray) and SUMO2K11Q (purple). The relative abundance (y-axis) of the indicated SUMO-SUMO2 linkage sites (x-axis) are shown. The measured intensity for K11 in the SUMO2WT under control conditions was set to 100% and the relative abundance for the individual residues was calculated in relation to this value. One of two independent replicates is shown. (d) Model depicting the possible impact of an acetylation of SUMO2 at K11 on formation of SUMO signaling. SUMO2/3K11Ac can restrict chain length by acting as a K11 chain terminator (A), SUMO2/3K11Ac can function as a binding platform for acetyllysine binding proteins (B), SUMO2/3K11Ac can trigger the formation of alternatively linked SUMO2/3 chains (C).
Figure 5A. Immunohistochemistry analysis of FATE1 expression in normal adrenal cortex and adrenal hyperplasia, adenoma and carcinoma (ACC). Testis tissue was used as positive control. Scale bars, 200 µm (low magnification); 50 μ m (high magnification).B. Table summarizing the relative intensity of FATE1IHC staining (H-score) in groups of normal, benign and malignant tissues. The highest FATE1 expression levels were found in ACC. n, number of samples studied for each category of tissues. NBST, normal and benign steroidogenic tissues. C. Prognostic parameters in ACC patients. Results are presented as hazard ratios (HR) including 95% confidence interval (CI). The significance level was set at α=5% for all comparisons. The results for SF-1, FATE1 and combined SF-1/FATE1 expression levels are shown both in univariate and mutivariate (adjusted for age, sex and ENSAT tumour stage) analysis. D. Overall survival of ACC patients according to FATE1 expression in their tumours, as measured by quantitative immunohistochemistry (low, green curve; high, red curve) in mutivariate analysis. p=0.008. E. Diagram illustrating FATE1 effect on ER-mitochondria distance and Ca2+ influx into mitochondria. In the presence of FATE1 (right) at the level of MAM, coupling between ER and mitochondria and Ca2+ influx into mitochondria decrease, with consequent resistance to apoptosis triggered by oxidative stress and chemotherapeutic drugs.
Figure 5T cells were purified from splenocytes of C57BL/6 mice by magnetic separation. The cells were cultured on plates pre-coated with (A) anti-CD3 antibody (1 µg/ml) or (B) anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies in the presence of graded doses of hTAPBPL-Ig (10 and 15 µg/ml) or equimolar amounts of control Ig (3.75 and 5.63 µg/ml) for 3 days. [3H] thymidine (1 µCi/well) was added to the cultures 12 hours before harvest. T cell proliferation was measured by [3H] thymidine incorporation. (C-E) Splenic cells were labelled with CFSE and cultured in 96-well plates that were precoated with anti-CD3 antibody and hTAPBPL-Ig or control Ig for 3 days as in (A). The cells were analyzed for CFSE levels by CD4+ and CD8+ T cells. Representative flow cytometric analysis of CFSE distribution of CD4+ and CD8+ T cells, Splenic cells were labelled with CFSE and cultured in 96-well plates that were precoated with anti-CD3 antibody and hTAPBPL-Ig or control Ig for 3 days as in (A). The cells were analyzed for CFSE levels by CD4+ and CD8+ T cells. statistical analysis of (D) CD4 and (E) CD8 T cell proliferation. Purified human T cells were cultured with plate-bound anti-human CD3 antibody (1 µg/ml) in the presence of graded doses of hTAPBPL-Ig (1.5 and 3 µg/ml) or control Ig protein (1.5 and 3 µg/ml) for 3 days. Cell proliferation was measured by [3H] thymidine incorporation. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.
Reduced tumor cell growth upon loss of ADAR1 is dependent on LGP2.Figure 5A) Kaplan-Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer (BLCA; n=407), breast cancer (BRCA; n=1099) and sarcoma (SARC; n=263) TCGA datasets. Median cut-offs were used for patient stratification and logrank test P values are shown. B) CAL27 cells transduced with doxycycline-inducible shRNAs targeting ADAR1 or GFP (negative control) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 (siLGP2 #1 or #2) or a control siRNA (siCtrl). To determine cell confluency at 120h post-transfection, cells were fixed, stained with Crystal Violet, and imaged. Images of a representative experiment are shown (n=3). C) Crystal Violet was extracted from stained cells (B) and the dye intensity was quantified using a colorimetric assay (OD590). OD590 values of doxycycline-treated cells were normalized to the OD­590 values of untreated cells. Quantification of data from three independent experiments is shown as mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.
SILAC phosphoproteomic approach for the identification of PINK1‐dependent targets.Figure 1Illustration of SILAC phosphoproteomics workflow. Flp‐In T‐Rex HEK293 cells stably expressing FLAG alone were cultured in unlabelled (R0K0) medium, kinase‐inactive (KI; D384A) PINK1‐FLAG cells were "medium" (R6K4) labelled, and wild‐type (WT) PINK1‐FLAG cells were "heavy" (R10K8) labelled using SILAC media containing the respective isotopes. All conditions were treated with 10 μM CCCP for 3 h and subjected to membrane fractionation. Four biological replicates of the samples were mixed in a 1:1:1 ratio, digested and fractionated by HILIC. Phosphopeptides from these fractions were enriched by TiO2 chromatography and analysed by quantitative mass spectrometry on an Orbitrap Velos Pro mass spectrometer. Data were analysed by MaxQuant and Perseus software packages. About 25 μg of membrane‐enriched lysate from the mass spectrometry experiments was immunoblotted with anti‐PINK1 antibody. TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm, respectively.
Figure 3(A) 3D structure of the Vav1 CSH3 domain. The previously described intramolecular inhibitory sites 1 and 2 of the Vav1 CSH3 domain are depicted in orange and yellow, respectively The regulatory Y836 phosphosite is highlighted in blue (Barreira et al., 2014). The residues present in the new inhibitory interface of the CSH3 are shown in raspberry color. (B) 3D structure of the VAV1 SH2 (red) bound to a Syk tyrosine-phosphorylated peptide (yellow). The mutated residues are shown in purple. p-Y, tyrosine phosphorylated. (C) Levels of phosphorylation of the Y174 phosphosite of the indicated Vav1 mutant proteins immunoprecipitated from Jurkat cells (upper panel). The total amount of Vav1 immunoprecipitated in each sample is shown in the bottom panel (n = 3 independent experiments).
Figure 7Representative images showing cells with ectopic expression of LATS2, which is labelled with GFP (the control is empty vectors). Relative mRNA levels of LATS1, LATS2, and YAP in the control and LATS2-expressing hOSEs at the 4th passage. Relative mRNA levels were determined with RT-PCR. Growth curves of hOSE cells at the 4th passage with or without ectopic expression of LATS2. Representative images showing the effects of LATS2 overexpression on the morphology and growth of hOSE cells at the 9th passage. Growth curves of control and LATS2-overexpressing hOSE cells. Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 4th passage with or without ectopic expression of LATS2. Representative images showing the effect of LATS2 on the morphology and β-galactosidase activities in cultured primary hOSE cells at the 9th passage. Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 9th passage with or without ectopic expression of LATS2. mRNA levels of major components of the DREAM complex (E2F4, E2F5, RBBP4, TFDP2) in the control and LATS2-overexpressing hOSEs at the 9th passage. Relative mRNA levels were examined by real-time PCR. Growth curves of the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells at their 16th passages. Representative images showing cellular morphology and β-galactosidase activities in control and LATS2-overexpressing hOSEs (K). Scale Bar: 50 µm. Right bar graph (L) showing quantitative results of SA-β-gal positive cells in the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells.
Figure 5A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with indicated dose of KY-02327 for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3] B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot. B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot (B) or immunofluorescence (C) analyses. Nuclei were counterstained with DAPI (C, blue). D. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 14 days. The mRNA level of collagen 1a (Col1a) was measured by quantitative real-time qRT-PCR. [n=3] E. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 21 days. The mRNA level of osteocalcin (OCN) was measured by qRT-PCR. [n=3]
DOR loss‐of‐function reduces autophagy.Figure 4(A,B) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled RNA, DOR siRNA1 or DOR siRNA2. Endogenous LC3 was detected by immunofluorescence. LC3‐positive puncta were counted in 100 cells per group under basal conditions or starved for 20 min. Scale bars, 10 μm. *Significant effects of starvation, P0.001; τsignificant effects caused by DOR knockdown, P0.001. (C,D) Scramble or DOR siRNA1 C2C12 cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes. Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen cells. *Significant effects of DOR repression, P0.001. (E) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA. *Significant effects of DOR knockdown, P0.01; τsignificant effects caused by 3MA, P0.01. 3MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
c to form a stable complex in solution A: Chromatogram and SDS-PAGE analysis of elution fractions from analytical size exclusion chromatography.Figure 1A: Chromatogram and SDS-PAGE analysis of elution fractions from analytical size exclusion chromatography. Dam1c and Bim1 individually or in combination were subjected to gel filtration. Bim1 coelutes with Dam1c and forms a stoichiometric complex. B: Lysine-lysine proximity map of Dam1c-Bim1 complex crosslinked by BS3. Each protein is represented as bar. Lines between the different proteins represent a pair of crosslinked lysine residues identified by mass spectrometry. Subunits of Dam1c that were crosslinked with Bim1 are shown in red, all other subunits in blue and Bim1 in green. The positions of an SxIP sequence in the C-terminus of Duo1 and Q205 in the C-terminus of Dam1 are marked by vertical lines. C: Comparative structural analysis of representative 2D class averages of Dam1WTc (upper row) and Dam1WTc-Bim1 (lower row). Densities exclusive to Dam1WTc (arrow) indicate the topology of Bim1 (scale bar; 10 nm). D: Three-dimensional structures of Dam1WTc (blue) and Dam1WTc-Bim1 (yellow) computed from the 2D class averages. The location of the protrusion domains of the Dam1WT complex (composed of Spc19/Spc34) is indicated (gray dashed ovals). Additional density in the Dam1WTc/Bim1 reconstruction (yellow dashed oval) indicates binding of Bim1 to the protrusion domains of Dam1c. E, F: EM images of negatively stained Dam1c alone (E) and in the presence of Bim1 (F), at low salt conditions. The final concentrations of Dam1c and Bim1 were 0.3 µM and 0.1 µM, respectively. The scale bars are 100 nm, boxes mark representative complexes shown as magnified inserts.
Figure 3A. Western blot detection of p-MST1, MST, p-YAP, YAP and TAZ in control and 40 h following PH in young and aged mice. TBP was used as a loading control. Representative results of a single experiment with n = 6 independent animals. Each band is from an independent animal. Western blot quantification ratios are graphically depicted. Proteins levels were normalized to TBP and mean value of the group is plotted. Unpaired, two-tailed Student t-test was used to calculate the significance between 0h (resected liver) and 40h following PH. Error bars represent mean ± SD B. RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, Cyclin A2, Cyclin D1 and p21. RT-PCR data are presented as log2 fold change and each value post PH was calculated for each mouse by comparison to its resected liver lobe. Circle, with red outline marks the aged non-regenerating livers shown in Figure 2B. Each dot is representing an independent animal.
Figure 3A. Middle. Lysates from 3xFLAG-OFD1 overexpressing HEK293 cells were added to beads with immobilized GST-fused LC3B, LC3BF52A-V53AdG, dNLC3BdG and GST alone. Right. Lysates from 3xFLAG-OFD1(OFD1) and empty vector (Empty) overexpressing HEK293 cells were added to GST-GABARAP-L1 and GST. Left. WB of total lysates (input). All panels display WB for FLAG, ACTIN is the loading control. B. Representative confocal images of endogenous OFD1 (green) co-staining with LC3B (red), and of 3xFLAG-OFD1 (red) with GABARAP (green) in HK2 cells (HBSS,90min). Hoechst labels nuclei (blue). Insets show magnifications and single-color channels of selected areas. Scale bar=10μm; n≥50 cells/sample. C. OFD1 domains architecture and alignment of LIR motif (boxed) with orthologous sequences. Residues with full (*), strong (:), weak (.) conservation are indicated. LisH=Lis Homology domain, CC=Coiled coil, LIR=LC3 interacting region. D. Lysates from 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1∆LIR(OFD1∆LIR) and empty vector (Empty) overexpressing cells were added to GST-GABARAP-L1, GST-LC3B and GST. E. Representative confocal images of OFD1∆LIR co-staining with LC3B (left) and GABARAP (right) in HK2 cells (HBSS,90min). Green, LC3B and GABARAP; red, 3xFLAG-OFD1∆LIR; blue, Hoechst for nuclei. Scale bar=5μm. n≥50 cells/sample.
Figure 11Oct4-eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), Cells were co-infected with 4 reprogramming factors (4F= Oct4, Sox2, Klf4, c-myc = OSKM) and double-infected cells were FACS sorted for analysis. (A) RT-qPCR analysis on day-4 after infection to determine RNA expression of Tnfaip2 or Vim in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test]. Oct4-eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), (B) RT-qPCR analysis on day-4 after infection to determine RNA expression of Vim or Tnfaip2 in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test]. Oct4-eGFP reporter MEFs or WT MEFs were (A-D) infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), ]. (C) Representative images of FACS profiles and (D) quantification of SSEA1+ iPSCs on day 14 after infection of MEF cultures with the indicated combination of shRNAs plus cDNAs (OE) or scrambled shRNA control [n=3 independent cultures per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-way ANOVA and Dunnett's multiple comparison test ] Oct4-eGFP reporter MEFs or WT MEFs were (E,F) infected with the indicated shRNA with or without combined treatment with Etomoxir - a chemical inhibitor of CPT1A. (E) Representative FACS profiles and (F) quantification of the percentage of SSEA1+ iPSCs in MEF cultures on day-18 after infection with shRNAs targeting Tnfaip2 or a scrambled shRNA control with or without continuous treatment with Etomoxir [n=5 biological replicates; data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons].
Listeria induces a transient activation of host AA starvation pathways.Figure 1(A) HeLa cells were infected with Listeria for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies. (B) HeLa cells left unstimulated (CTR) or infected with Listeria for 1 or 3 h, analysed by IF using antibodies against mTOR and LAMP2. (C) Percentage of Listeria‐infected cells displaying mTOR localization to LAMP2+ vesicles. Values are means±s.e.m. n=3. ***P0.001 and **P0.01 over uninfected. (D, E) HeLa cells were infected with Listeria for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies. (F) qPCR analysis of ATF3 induction following infection of HeLa cells with Listeria for 1-4 h. Values are means±s.e.m. n=3. Scale bars: 10 μm.Source data for this figure is available on the online supplementary information page.
RNA secondary structure of SARS-CoV-2 miR-O7a precursor and sequence conservation among different SARS coronaviruses and within SARS-CoV-2 variants.Figure 2(A) Predicted RNA secondary structure for the CoV2-miR-O7a and flanking sequence using the first 70 nt of the open reading frame of the ORF7a. The arrows indicate the sites of the miRNAs possibly cleaved by Dicer. The stem-loop structure is not conserved in SARS-CoV. The colors indicate the base-pair probabilities. (B) Conservation of the first 70 nt of the ORF7a sequence among different SARS coronaviruses. The underlined sequences are related to the position of the SARS-CoV-2 miR-O7a. The conserved ribonucleotides of the CoV2-miR-O7a sequence are marked in red and in blue all the non-conserved ribonucleotides across the 70nt sequence. The bat and pangolin coronaviruses closely related to SARS-CoV-2 are marked in purple. (C) Percentage of conservation along the nucleotide positions in the ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. The first 70 nt are shown in red and shows a higher percentage of conservation compared to the rest of the sequence of ORF7a. Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71-366 nt of ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. Box plots display median (line), first and third quartiles (box), and 5th /95th percentile value (whiskers). Each dot represents the outliers. Two-tailed P values were calculated using the student's t-test.
The phenotype of Phb2pko mice is not developmental.Figure 3A PAS staining of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).B Immunohistochemistry for podocin on kidney sections of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm). C Coomassie stain of urinary samples of tPod-Phb2pko and control mice.
Fig.Figure 2A: Brightfield and fluorescence images of a NTH/GFP zygote at 4h post-fertilization, showing dispersed extranuclear fluorescence. Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm. B: Image of an NTH/GFP ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows). Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm. C: Schematic diagram of a genetic cross between parasite lines NTH/GFP and LAP3/mCherry and the resultant ookinete population after mosquito transmission and drug selection (oocysts, liver and blood stage parasites not shown). Colours represent NTH::GFP (green), LAP3::mCherry (red) and their co-localisation (orange). Spots in ookinetes depict crystalloids. Self-fertilization events are omitted. D: Brightfield and fluorescence images of a zygote at 4h post-fertilization simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red). Scale bar = 5μm. E: An ookinete simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red), showing co-localization in the crystalloid. Scale bar = 5μm. F: Western blot of purified NTH/GFP ookinete samples with (+) and without (-) prior in vivo crosslinking. Mr markers (kDa) are indicated on the right-hand side.
Mfn2 regulates PERK activity.Figure 7(A) Immunodetection of p‐PERK and p‐eIF2α in Mfn2 KO MEFs, Mfn2 knockdown 3T3‐L1 fibroblasts, and Mfn2 knockdown C2C12 myoblasts. (B) Densitometric quantification of p‐PERK and p‐eIF2α in MEFs. Data are mean±s.e.m. (n=3). *P0.05 versus WT cells. (C, D) Immunodetection of p‐eIF2α, eIF2α, CHOP, XBP‐1s, LC3b, and p62 in Mfn2‐deficient liver (C) or skeletal muscle (D) from tissue‐specific KO mice. (E) Co‐immunoprecipitation of Mfn2 and PERK in WT or Mfn2 KO cells stably expressing PERK‐myc or an empty vector (pBABE‐myc; negative control). (F) Co‐immunoprecipitation of endogenous Mfn2 and PERK in WT MEF lysates. PERK was immunoprecipitated with a C‐terminal antibody and co‐immunoprecipitation of Mfn2 was detected by western blot.Source data for this figure is available on the online supplementary information page.
RsaI inhibits glcU_2 and fn3K mRNA translation.Figure 3(A) Toe-print assays showing the effect of RsaI on the formation of the ribosomal initiation complex of glcU_2 and fn3K mRNAs, respectively. Lane 1 : incubation control of mRNA alone; lane 2 : incubation control of mRNA with 30S subunits; lane 3: incubation control of mRNA with RsaI; lane 4 : formation of the ribosomal initiation complex containing mRNA, 30S and the initiator tRNAfMet (tRNAi); lanes 5 to 9 : formation of the initiation complex in the presence of increasing concentrations of RsaI, respectively : 50 nM (lane 5), 100 nM (lane 6), 150 nM (lane 7), 300 nM (lane 8), and 400 nM (lane 9). Lanes T, A, C, G: sequencing ladders. The Shine and Dalgarno (SD) sequence, the start site of translation (START) and the toe-printing signals (+16) are indicated. At the bottom of the gels are shown the predicted interactions between RsaI and its targets. Translation start codons are in green, and the Shine and Dalgarno (SD) sequence is underlined, the arrowheads depict the toe-printing signals. (B) The β-galactosidase activity (Miller Units) has been measured from various fusions expressed from a plasmid which also carries rsaI gene under its own promoter: PrpoB::glcU::lacZ::rsaI, PrpoB::fn3K::lacZ::rsaI, PrpoB::treB::lacZ::rsaI, PrpoB::HG001_1242::lacZ::rsaI, and PrpoB::HG001_2520::lacZ::rsaI expressed in the mutant strain HG001-∆rsaI. The same constructs were made in the absence of rsaI gene. For the fn3K-lacZ fusion, we also used an additional construct PrpoB::fn3K::lacZ::rsaImut4 expressing both the fusion FN3K-LacZ protein and RsaI mut4. The β-galactosidase activity was normalized for bacterial density and the results represented the mean of four independent experiments. The error bars are standard deviations and the statistical significance was determined using the Student's t-test. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001, ns is for not significant.