EMBO/SourceData huggingface dataset:
Figure 6 (A Measurement of serum indices including liver enzymes (AST, ALT and GGT) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).  B) Measurement of kidney markers (CREA and UA) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).  (C) Morphological examination of liver and kidney from wild-type C57BL/6 (C57), untreated mdx controls (mdx) and treated mdx mice (scale bar: 100 μm).  (D Immunohistochemistry of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).  E) quantification of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).
Figure 1A. Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons (DIV16) expressing GFP-Sec61β. Zooms represent (1) an axonal structure with presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 µm (full size) and 5 µm (zoom).C. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).D. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N=2, n=288-541 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.E. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal.F. Pull down assay of HEK293T cells co-expressing Myc-VAPA with BioGFP or BioGFP-SCRN1. G. Pull down assay of HEK293T cells co-expressing Myc-VAPB with BioGFP or BioGFP-SCRN1.Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.H. Scaled representation of SCRN1-associated proteins identified with pull-down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP-SCRN1 from HEK293T cell lysates with adult rat brain extracts. All candidates showed >10 enrichment of PSM compared to control.I. Endogenous localization of SCRN1 and Syt in cortical neurons (DIV18) expressing GFP. Zoom represents an axon structure with presynaptic boutons (arrowheads). Scale bars: 10 µm (full size) and 5 µm (zoom).J. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).K. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA alone or SCRN1 shRNA with GFP-SCRN1. N=2, n=201-300 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.
Figure 2A. Localization of GFP-SCRN1 and TagRFP-ER in hippocampal neurons (DIV16-18). Scale bars: 10 µm (bottom panel, full size) and 5 µm (top panel; bottom panel, zoom).B. Localization of GFP-SCRN1 and ssRFP-KDEL in COS7 cells. Scale bars: 10 µm (full size) and 5 µm (zoom).C. Hippocampal neurons (DIV16) co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).D. COS7 cells co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).F. COS7 cells co-expressing HA-VAPB with GFP-SCRN2 or GFP-SCRN3. Scale bars: 10 µm (full size) and 5 µm (zoom).
Figure 3B. COS7 cells co-expressing GFP-SCRN1 with HA-VAPB-K87D/M89D. Scale bars: 10 µm (full size) and 5 µm (zoom).C. COS7 cells co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).D. Hippocampal neurons (DIV16) co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).E. Quantifications of SCRN1 recruitment to VAPB-positive structures in COS7 cells (%). Left graph: co-expression of GFP-SCRN1 with HA-VAPB or HA-VAPB-K87D/M89D ( N=2-3, n= 44-46). Right graph: co-expression of HA-VAPB with GFP or GFP-SCRN1, GFP-SCRN1-N, GFP-SCRN1-C, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A (N=2-3, n=41-64). Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Chi-square test with post-hoc analysis including Bonferroni correction.F. Pull down of HEK293T cells co-expressing Myc-VAPB with GFP, GFP-SCRN1, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A.
Figure 4B. Live hippocampal neurons (DIV17-18) co-expressing TagRFP-ER with GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Scale bars: 5 µm (full size) and 2 µm (zoom).C. Time-lapse of TagRFP-ER dynamics in hippocampal neurons (DIV16-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Intact ER structures (dark arrowheads), intact and dynamic ER structures (light arrowheads) and impaired an non-dynamic ER structures (arrows) are indicated. Scale bars: 5 µm (full size) and 2 µm (zoom).E. ER nanostructures visualized with GFP-Sec61β in axons of hippocampal neurons (DIV18) immunostained for α-Tubulin and co-expressed with pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs, and subjected to expansion microscopy. Scale bars: 2 µm (full size) and 500 nm (zoom).F. FRAP experiment of TagRFP-ER in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. Scale bar: 2 µm.G. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. H. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.I. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. J. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.
Figure 1B. Flow cytometry for Bu-1a expression in wild type cells with (GAA)n tracts of different length knocked into the BU-1A locus (in blue). DT40 cells are heterozygous and carry one BU-1A and one BU-1B allele. All experiments introducing repeats into BU-1A are carried out in cells in which the +3.5 G4 has been deleted from both A and B alleles, to avoid transvection between the allele Black outline: positive control (wild type cells); red outline: negative control (cells carrying a genetic disruption of BU-1)C, D. Bu-1a fluctuation analysis of wildtype and primpol cells in which the endogenous +3.5 G4 has been deleted (ΔG4) or with (GAA)10 and (GAA)20 sequence orientated such that it is replicated as the leading (C) or lagging (D) strand template for a fork entering from the 3' end of the locus as shown in panel (A). At least two independent fluctuation analyses were performed. Circles represent the percentage of Bu-1a loss variants in at least 24 individual clones from these experiments, with mean ± SD reported. ****p < 0.0001, * p ≤ 0.05, ns = not significant; one-way ANOVA.
Figure 2Human PrimPol, or mutants, tagged with YFP were expressed in primpol cells harbouring (GAA)10 sequence in the BU-1A locus. Bu-1a and YFP double positive cells were sorted and expanded for 2 weeks, and then analysed for Bu-1a expression variants. For each complementation, at least two independently derived clones were subjected to fluctuation analysis. As previously observe expression of hPrimPol[AxA] and hPrimPol[ZfKO] is deleterious and unstable. Cells expressing these mutations and remaining YFP-positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis. Pooled results from at least three independent fluctuation analyses are represented with mean ±SD indicated with red bar and whiskers. Statistical significance: **** p < 0.0001, *** p<0.001, * p ≤ 0.05, ns = not significant; Kruskal-Wallis test
Figure 3A. DRIP-qPCR analysis reveals accumulation of R-loops across the BU-1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H-treated samples and was normalised to -0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t-test was used to compare differences between matched amplicons in primpol BU-1A(GAA)10 and the other cell lines indicated. **** p ≤ 0.0001, ns = not significantB. DNA:RNA hybrids in primpol BU-1A(GAA)10:GgRNase H and primpol BU-1A(GAA)10:hPrimPol. An unpaired t-test on three biological replicates was used to compare differences to primpol BU-1A(GAA)10 for each matched amplicon. The bar represent the mean and whiskers the SD. *** p ≤ 0.001, ** p ≤ 0.01, ns = not significantC. Overexpression of chicken RNase H1 prevents (GAA)10 induced BU-1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU-1A (GAA)10 clones. One-way ANOVA was used to calculate the significance of differences in BU-1 instability between primpol BU-1A ΔG4 and other cell lines. **** p ≤ 0.0001, ns = not significantD. Diagram of the RNase H1 hybrid binding domain (HBD) - mCherry fusion and flow cytometry expression profiles of the construct in four clonesE. R-loop stabilisation induces epigenetic instability of BU-1. Bu-1a fluctuation analysis of wild type cells expressing HBD-mCherry. The scatter plots pool results from at least two different clones with matched HBD-expression. Mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant; one-way ANOVA.
Figure 4A . Expression of geminin-tagged chicken RNase H1-YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 (X-axis). RNase H1-YFP with or without the geminin degron protein is detected on the Y-axis. The RNase H1-YFP-geminin degron is degraded in G1. In contrast, RNase H1-YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replicationB. Bu-1a fluctuation analysis of two independently derived primpol BU-1A(GAA)10: Gg RNase H1-YFP-geminin degron clones. Since the expression of the RNase H1-YFP-geminin degron construct is not stable (unlike the RNase H1-YFP construct without the degron), Bu-1a expression was assessed separately in the YFP +ve and YFP -ve cells within each clone. Statistical differences calculated the Kruskal-Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significantC. DRIP-qPCR for R-loops around the engineered +3.5 (GAA)10 repeat in BU-1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU-1 DRIP signal was normalised to -0.5 kb amplicon in G1-arrested cells (t = 0 h) Black: wild type; red: primpol. Error bars = SD. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05E. Validation of analysis of nascent DNA:RNA hybrid formation in BU-1 locus. Enrichment of 4SU labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** p ≤ 0.01, * p ≤ 0.05, ns = not significant; unpaired t-testG. Newly synthesised R-loops in BU-1 during S phase in wild type (black) and primpol (red). Error bars represent 1SD of three biological repeats of the experiment. *** p ≤ 0.001, * p ≤ 0.05; unpaired t-test
Figure 5A. Representative normalised DRIP-seq data in two genes COL22A1, spanning over 200 kb, and MYC. The locations of H-DNA and G4 motifs are shown below the gene map. Wild type in blue; primpol in red. The corresponding RNAse H-treated samples are dashed. See Methods for further details of graphic generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).C. DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike-in. n (wild type) = 41445; n (primpol) = 48648. D. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). F. Normalised DRIP peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).G. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). H. Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).
Figure 6A. Representative normalised RNA DIP-seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H-DNA and G4 motifs are shown below the gene map. The corresponding RNAse H-treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).C. RNA DIP-seq peak heights in wild type and primpol BOBSC cells normalised to a DT40 spike-in. n (wild type) = 32740; n (primpol) = 33721. D. Correlation of normalised RNA DIP-seq peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).F. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).G. Normalised RNA DIP-seq peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).H. Normalised RNA DIP-seq peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).
Figure 1A Immunohistochemistry was used to assess STAMP2 expression in normal and malignant human prostate specimens. Tissue microarrays (TMAs) with normal prostate (n = 27) and early-stage (Gleason grade < 7) (n = 35) or late-stage prostatetumors (Gleason grade ≥ 7) (n = 149) were subjected to immunohistochemistry as described in Materials and Methods. Representative images are shown.B Quantification of cytosolicSTAMP2 staining in TMAs is shown in (A). One-way analysis of variance (ANOVA) with a post hoc test, *P < 0.0001. Error bars indicate SEM.C, D The details of cytosolic (C) and membrane (D) STAMP2 staining in the TMAs described in (A).
Figure 2A LNCaP cells were transfected with either control siRNA or two independent STAMP2 siRNAs, and membrane fractions of the cells were prepared followed by Western blotting analysis. STAMP1 is used as a loading control.B LNCaP cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h before being transfected with the indicated siRNAs. The cells were then cultured for the indicated times, and cell growth was measured by Cell Counting Kit-8. n = 3, *P < 0.0001. Error bars indicate SD.C LNCaP cells were cultured and transfected with the indicated siRNAs as described in (B). After transfection, the cells were cultured for 10 days. The colonies formed were stained and photographed.D LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881. The cells were then collected, and membrane fractions were prepared followed by Western blot analysis.E LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured for the indicated times, and the cell numbers were determined using a hemocytometer. n = 3, *P = 0.047; **P = 0.0046. Error bars indicate SD.F LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 weeks as described in Materials and Methods. The plates were then stained and photographed.G Quantification of data from (F). n = 3, *P = 0.022. Error bars indicate SD.F LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 weeks as described in Materials and Methods. The plates were then stained and photographed.G Quantification of data from (F). n = 3, *P = 0.022. Error bars indicate SD.H LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were subcutaneously implanted into male SCID mice. Tumor size was measured after 8 weeks. n = 9, *P = 0.037. Error bars indicate SEM.I VCaP cells were transfected with the indicated siRNA as described in (B). After transfection, VCaP cells were cultured for 2 weeks. The colonies formed were stained and photographed.J Quantification of the data from (I). n = 3, *P < 0.0001. Error bars indicate SD.
Figure 3A LNCaP cells were transfected with either control or STAMP2-specific siRNA. Two days after transfection, cells were subjected to cell cycle analysis as described in Materials and Methods. Representative histograms showing PI-stained cells are shown.B The proportion of cells in each stage of the cell cycle from the experiment in (A) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.0002; **P = 0.0056; ***P = 0.0099. Error bars indicate SD.C Two days after transfection with the indicated siRNAs, LNCaP cells were harvested and cell lysates were prepared and subjected to Western blot analysis with the indicated antisera. Representative blots of three independent experiments are shown.D LNCaP cells were transfected with either control or STAMP2-specific siRNA. Three days after transfection, cells were treated with either 50 ng/ml TRAIL or 20 μmol/l LY294002 (LY) for 24 h, or both agents for 6 h, and then subjected to TUNEL/FACS analysis. Representative histograms of TUNEL-stained cells are shown. FL1-H refers to the gating of the cells for the TUNEL staining with fluorescence measurement.E The extent of apoptosis from the experiment in (D) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.027; **P = 0.0058; ***P = 0.0016. Error bars indicate SD.F LNCaP cells were transfected with either control or two independent STAMP2-specific siRNAs, ST2-1 and ST2-2. Three days after transfection, cells were treated in the same way as in (D). Whole-cell lysates were prepared and subjected to Western blot analysis with the indicated antisera.G LNCaP cells stably expressing either control or STAMP2 vector were treated in the same way as in (D). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies.
Figure 4A STAMP2 expression was determined by IHC of a neoadjuvant hormone therapy (NHT) TMA containing samples from hormone naïve (untreated) (n = 28), NHT 1-6 months (n = 31), NHT 7-12 months (n = 41), and patients with PSA recurrence (n = 21) as indicated. Representative images are presented.B Quantitative presentation of the data from (A). Unt, untreated; Recur, PSA recurrence. The Kruskal-Wallis test was used for significance analysis. *P = 0.0499. Error bars indicate SEM.C Boxplots of STAMP2 expression levels in normal prostate tissue, primary prostate cancer, and metastatic CRPC were analyzed in two independent GEO datasets (GSE6919 and GSE35988). The values of individual samples are presented as dots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles. The statistical significance, indicated in the tables at the bottom, was determined by Student's t-test.D CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. mRNA was extracted from the tumors and used for qPCR analysis of STAMP2 expression. The results are presented as boxplots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles, and the outlier is presented as an open circle. The statistical significance was determined by one-way ANOVA with a post hoc test. n = 3 in group week 0, 1, 2, and 4; n = 5 in refractory group. *P = 0.011; **P = 0.049. Error bars indicate SEM.