EMBO/soda-nel huggingface dataset:
HDAC inhibitors tune miRNAs in Extracellular Vesicles of Dystrophic Muscle-Resident Mesenchymal Cells (2020)
Figure 3

Fig. 3: HDACi increase miR-206-3p amounts in EVs from DMD FAPs.

(A) Representative images of Sca-1 (green), Laminin (white), DAPI (blue) immunofluorescence, and miR-206-3p (violet) immunohistochemistry on 10 uM sequencial cryosections of Tibialis Anterior (TA) of young mdx mice (CTR) and after TSA treatment (TSA) (0.6 mg/kg/day for 15 days by i.p.) (n=6). Scale bar = 25 μm.

(B) Graph showing the quantifications of miR-206-3p violet area relative to conditions indicated in (A). Star (*) indicates t-test analysis **p<0.01.

(C) Graph representing the miR-206-3p relative expression in EVs isolated from muscle interstitium of tibialis anterior from wild type mice (CTR), mdx young and old mice (control -CTR- and TSA treated) (n=4). Star (*) indicates Tukey analysis compared to wild type mice (CTR), ****p < 0.0001; hash (#) indicates Tukey analysis compared to mdx young CTR, #### p<0.0001. § represents statistical analysis by Anova test. §§§§ p<0,0001.

(D) Representative images of CD90 (green), Dapi (blue) and miR-206-3p (violet) immunohistochemistry in brachial biceps bioptic samples of DMD patient before (CTR) and after Givinostat treatment (GIV). Scale bar = 50 μm.

(E) Graph showing the quantifications of miR-206-3p violet area measured as pixel^2/field relative to the experimental points indicated in (D) (n=18, biological replicates). Star (*) indicates t-test analysis. **p<0.01.

(F) Graph showing the relative expression of miR-206-3p in EVs isolated from a human population of muscle resident cells enriched in FAPs (ehFAPs) isolated from biopsies of two DMD patients (DMD-1 and DMD-2) before (CTR) and after (GIV) Givinostat treatment, Two technical replicates are shown.

Data information: Nuclei were counterstained with DAPI (blue). All data for (B, C and E) correspond to the average ± SEM while the data for (F) correspond to the average ±SD.
Chronic miR-29 antagonism promotes favorable plaque remodeling in atherosclerotic mice (2016)
Figure 2

Figure 2: LNA-miR-29 increases fibrous cap thickness and SMA staining and reduces necrotic zones in lesions

(A) Movat pentachrome staining of aortic root (upper panel-4x magnification) and brachiocephalic artery fibrous caps (lower panel-10x magnification) at 14 week harvest, caps denoted with arrowheads and black bars (roots only). (B) Quantification of aortic root (left panel, *p=0.005, #p<0.001) and brachiocephalic artery (right panel*p=0.011, #p<0.024) fibrous caps, graphed as percent of total plaque area. (C) Representative immunofluorescence (IF) staining of smooth muscle actin (red), denoted by white arrowheads, in aortic root lesions shown at 10x magnification. IF images in (C) are representative of data quantified in (D) calculated from 3-6μm serial sections from animals in independent cohorts. Level of significance was determined using Kruskal-Wallace one-way ANOVA on ranks, *p=0.002 (D). (E) Movat pentachrome staining of aortic root (upper panel-4x magnification, *p<0.001) and brachiocephalic artery necrotic areas (lower panel-10x magnification) at 14 week harvest, representative necrotic zones denoted by highlighted areas. (F) Quantification of aortic root (left panel, *p<0.001) and brachiocephalic artery (right panel, #p=0.044) necrotic areas, graphed as percent of total plaque area. Data in (B, F) are averaged percentages per total plaque area from 4-6μm serial sections from each animal. All of the data represent the mean + SEM, *p<0.05 versus LNA-control tissue, # represents p<0.05 versus saline tissue, n=9, 13, 15 . Level of significance was determined using one-way ANOVA with Bonferroni’s post test.
Chronic miR-29 antagonism promotes favorable plaque remodeling in atherosclerotic mice (2016)
Figure 3

Figure 3: LNA-miR-29 increases collagen in murine plaques

ApoE-/- common carotid arteries (CCA) were dissected into plaque and adjacent non-plaque areas, then RNA isolated. (A) Graphed qPCR quantification of collagen 1A1 (left panel) and collagen 3A1 (right panel) mRNA expression in plaque (P) and non-plaque (NP) areas of ApoE-/- carotid arteries. Three assays were carried out, and the data represent mRNA expression normalized to NP, and the mean + SEM, n=4, 3, 4 of mice/group. * represents p<0.001 compared with LNA-miR-29 non-plaque region. Picrosirius red staining of collagen in BCs (10x magnification) (B); and aortic roots (4x magnification) (D); brightfield (upper panel) and polarized light (lower panel) imaging shown. Collagen quantification in BCs (C, *p=0.016) and roots (E, *p=0.003, #p=0.005) from polarized light images expressed as a percentage of plaque area. Data in (C, E) are averaged percentages per total plaque area from 4-6μm serial sections from each animal. All of the data (A, C, E) represent the mean + SEM, * represents p<0.05 versus LNA-control tissue, # represents p<0.05 versus saline tissue, n=9, 13, 15. Level of significance was determined using one-way ANOVA with Bonferroni’s post test.
Chronic miR-29 antagonism promotes favorable plaque remodeling in atherosclerotic mice (2016)
Figure 4

Figure 4: Modulation of miR-29 defines murine VSMC proteome and collagen targets

Proteome studies analyze the frequency distribution of protein changes after the transfection of primary murine aortic VSMCs with pre–miR-29b, anti–miR-29b, pre–miR-30c, or anti–miR-30c. A log2 fold change of −1 or 1 was used as cutoff. Log2 (fold change) is calculated using the normalized spectral counts and averaged for the 3 biological replicates. (A) The log (fold change) for differentially secreted proteins following overexpression or inhibition of pre–miR-29b was analyzed, and significantly changed miR-29 confirmed target transcripts are shown. FDR indicates false discovery rate. The complete list of significant proteins is in (Appendix Table S1A-B). (B) Thirty microliters of conditioned medium from VSMCs transfected with pre–miR-29b and anti–miR-29b were separated by SDS-PAGE before validation of COL3A1 by immunoblotting. (C) Densitometry analysis of COL3A1 signal from blot in (B) (pre-miR, left *p<0.001, anti-miR, right *p=0.002) . All data in (C) represent the mean + SEM, *p<0.05 versus other groups in anti- and pre- groups. Level of significance was determined using one-way ANOVA with Bonferroni’s post test. (D) Working model of LNA-miR-29 effects on plaque composition as detailed in the data of this paper. Administration of LNA-miR-29 blocks lesion growth and increases intra-plaque collagen expression and fibrous caps, two indices characteristic of more stable plaques. This panel was produced using Servier Medical Art (www.servier.com).
Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3 (2009)
Figure 1

(A) Immunoblot analysis of BAG1, BAG3, Hsc/Hsp70 and Hsp90 in young and old I90 cells. For detection of BAG proteins, an antibody directed against the conserved BAG domain was used. (B) Real‐time PCR analysis of human BAG family members and Hsc70 and Hsp90 in young and old I90 cells. Depicted is the expression ratio (log2) of target genes in old cells relative to young cells. *P0.05 and **P0.01 versus young, n=3. (C) Co‐IP analysis of Hsc/Hsp70 interaction with BAG1 and BAG3 in young (Y) and old (O) I90 cells. Upper panel shows relative amounts of proteins in cell lysates used for Co‐IP (Input). Middle panel shows levels of BAG1 and BAG3 found in Hsc/Hsp70 immunoprecipitates. Lower panel shows levels of Hsc/Hsp70 co‐sedimented upon immunoprecipitation of BAG3. (D, E) 293 cells were treated with the indicated agents for 8 h followed by immunoblot analysis of indicated proteins.
Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3 (2009)
Figure 2

(A) 293 cells were transfected with bag1, bag3 or nonsense (nons) siRNAs, as indicated. After 48 h, cells were transfected with d2GFP expression plasmid together with half the amounts of the indicated siRNAs. After additional 24 h, levels of indicated proteins were detected by immunoblot analysis. (B) 293 cells were transfected with indicated siRNAs for 48 h followed by real‐time PCR analysis of BAG1 and BAG3 mRNA levels. Depicted is the mean relative expression ratio (log2) ±s.e.m. *P0.05 and ***P0.001 versus nons, n=3. (C) BAG1 knockdown was performed in 293 cells for 48 h. Thereafter, cells were transfected with GFP or GFP‐based UPS reporter genes as indicated. After additional 24 h, levels of GFP‐positive proteins were detected by western‐blot analysis using a GFP antibody. (D, E) Immunoblot analysis of indicated proteins from d2HEK cells treated with different concentrations of MG132 for 5 h (D) or transfected with indicated amounts of BAG1S plasmid for 24 h (E).
Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3 (2009)
Figure 3

(A) 293 cells were transfected with nonsense (nons), bag1 or bag3 siRNAs for 48 h and then treated for 2 h with the lysosomal inhibitors pepstatinA and E64 (both 10 μg/ml; Pep.A/E64) or DMSO as control, followed by immunoblot analysis of the indicated proteins. (B) 293 cells were transfected for 48 h with the indicated siRNAs and BAG3 expression plasmid (BAG3‐N1) or vector control (N1) followed by the same analysis as in (A). (C) Diagram shows the autophagic flux of 293 cells with differently modulated BAG1 and BAG3 levels as described in (A) and (B). Autophagic flux was determined by the strength of LC3‐II accumulation in a 2‐h treatment period with Pep.A/E64. Therefore, normalised LC3‐II levels in the absence of inhibitors were subtracted from corresponding levels obtained in the presence of Pep.A/E64. Values are expressed as mean±s.e.m. *P0.05 versus control‐transfected cells, n=3. (D) I90 cells were transfected with GFP fused to LC3 (pGFP.LC3) and co‐transfected either with BAG3‐N1 or N1. After transfection for 48 h, cells were treated as in (A) and levels of indicated proteins were detected by immunoblot analysis. (E) I90 cells, transfected as in (D) for 24 h, were microscopically analysed for GFP‐LC3 fluorescence. Representative pictures are shown. Bar: 20 μm. Values expressed in diagram are mean±s.e.m. from three independent experiments (50 cells were counted per experiment). *P0.05 versus N1, n=3.
Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3 (2009)
Figure 4

(A) I90 cells were transfected with BAG3 (BAG3‐N1) or vector control. After transfection for 48 h, levels of indicated proteins were detected by western‐blot analysis. (B) Transcript levels of SQSTM1 in I90 cells transfected as in (A) were compared by real‐time PCR analysis. Depicted is the fold‐increase of SQSTM1 mRNA levels in BAG3‐N1‐transfected cells relative to control (N1)‐transfected cells. Values are expressed as mean±s.e.m. **P0.01 versus N1 cells, n=3. (C) I90 cells were transfected with a BAG3‐GFP fusion plasmid for 48 h followed by indirect immunofluorescence staining of endogenous SQSTM1. (a) Direct fluorescence of BAG3‐GFP (green), (b) indirect immunofluorescence of SQSTM1 (red) and (c) the stainings of (a) and (b) overlapped. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 10 μm. (D) Co‐IP studies testing the interaction of SQSTM1 and BAG3. I90 cells were transfected as in (A). After 24 h, SQSTM1 and BAG3 were immunoprecipitated followed by analysis of co‐sedimented proteins, as indicated. As negative control purified rabbit (rb) and mouse (ms) IgG was used. Left panel shows relative amounts of proteins in lysates used for Co‐IP (Input).
Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3 (2009)
Figure 5

(A) Proteasomal chymotrypsin and cathepsin activity in lysates from young (Y) and old (O) I90 cells was determined using specific fluorescence probes, as described in the Material and methods section. Values are expressed are mean±s.e.m. *P0.05 and **P0.01 versus young, n=3. (B) Real‐time PCR analysis of LC3, WIPI1 and SQSTM1 mRNA levels in young and old I90 cells. Depicted is the mean expression ratio (log2) ±s.e.m. of target genes in old cells relative to young cells. **P0.01 and ***P0.001 versus young, n=3. (C) Immunoblot analysis of LC3 and SQSTM1 in young and old I90 cells. (D) Young and old I90 cells were treated for 2 h with bafilomycin A1 (BafA1, 2 μM) or DMSO as control followed by immunoblot analysis of indicated proteins. (E) Immunoblot analysis of WIPI1 expression in young and old I90 cells. (F) Indirect immunofluorescence staining of endogenous LC3 (green), SQSTM1 (red) and WIPI1 (white) in I90 cells of young and old age. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells with indicated characteristics counted as in Figure 3E. (G) Old and young I90 cells were treated for 1 h with DMSO as control (C), lactacystin (L, 2 μM) or NH4Cl (20 mM) plus leupeptin (Leu, 5 μM) (N, NH4Cl/Leu). Western‐blot analyses were performed for detection of indicated proteins. In the diagram (right panel), levels of polyUb‐proteins and SQSTM1 are depicted after normalisation to corresponding Tubulin levels. Values are expressed as mean±s.e.m. *P0.05 versus old control, #P0.05 versus young control, n=3. (H) I90 cells of old (upper panel) and young (lower panel) age were transfected with atg7 or nonsense (nons) siRNA. After transfection for 4 days , the cells were treated with NH4Cl/Leu or DMSO for 1 h followed by fractioning of cell lysates in TritonX‐100 (TX‐100) soluble and insoluble material. Equal protein amounts of both fractions were directed to immunoblot analysis for analysis of indicated proteins. Gapdh and Histone H3 were used as loading controls of soluble and insoluble fractions, respectively. (I) Transmission electron microscopic analysis of young (a) and old (b) I90 cells. Magnifications of marked areas in (a) and (b) are shown in (c) and (d), respectively. An arrow indicates an autolysosome, and arrow heads indicate autophagosomes. M, mitochondrion. N, nucleus.
Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3 (2009)
Figure 6

(A) Immunoblot analysis of indicated proteins of young (Y) and old (O) I90 cells upon fractionation as described in Figure 5H. (B, C) After transfection for 96 h of old I90 cells with bag3 or nonsense (nons) siRNA, BAG1 and BAG3 protein and mRNA levels were analysed by immunoblot (B) and real‐time PCR (C) analysis, respectively. Transcript levels in bag3 siRNA cells are depicted as the mean log2 expression ratio ±s.e.m. relative to nons siRNA cells. *P0.05 and ***P0.001 versus nons, n=3. (D) Indirect immunofluorescence analysis of endogenous LC3 (green) and WIPI1 (white) in old I90 cells transfected with nons (a, b) and bag3 siRNA (c, d) for 96 h. DAPI (blue) was used as a nuclear marker. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells counted as in Figure 3E. (E) Same analysis as in Figure 5D but old cells transfected as in (A) were used. Values expressed in the diagram are mean±s.e.m. *P0.05 versus nons, n=3. (F) Same analysis as in Figure 5G but old I90 cells transfected as in (A) were used. C, control; L, lactacystin, N, NH4Cl/Leu. Values are expressed as mean±s.e.m. *P0.05 versus nons control, or as indicated, n=3. (G, H) Same analysis as in (F) but young I90 cells 48 h after transfection with a BAG3 expression plasmid (BAG3‐N1) or vector control (N1) together with nons or sqstm1 siRNA (G) and nons or atg7 siRNA (H), as indicated, were used. *, #, ∼ and +, P0.05 versus C N1, C BAG3‐N1, C BAG3‐N1 sqstm1 or atg7 and C N1 sqstm1 or atg7, n=3.
Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3 (2009)
Figure 7

(A) Protein extracts from cerebellum (CER) of young (Y, 3 months) and old (O, 24 months) mice were analysed for BAG1 and BAG3 expression by immunoblot analysis. (B) Real‐time PCR analysis of indicated mRNA levels in cerebellum of young and old mice. Depicted is the log2 expression ratio of target genes in old mice relative to young mice. Values are expressed as mean±s.e.m. *P0.05 and **P0.01 versus young, n=3. (C) Immunoblot analysis as in (A) but in cortex (CTX), hippocampus (HIP) and mid‐brain (MB). (D) Western‐blot analysis of indicated proteins in different brain regions of young and old mice. (E) Total cathepsin and specific cathepsin B activity in brain extracts from young and old mice was determined using fluorescence probes Z‐FR‐AMC and Z‐RR‐AMC, respectively, as described in the Material and methods section. Values are expressed as mean±s.e.m. *P0.05 versus young, n=3. (F) Expression analysis of indicated proteins in primary hippocampal astrocytic and neuronal cell cultures from young (2 months) and old (24 months) rats. Detection of GLT1 and NeuN served as astrocyte and neuron markers, respectively. Note the NeuN signals in astrocytic cultures. We attributed these signals to the presence of resting nuclei of dead neurons, which we observed under the microscope.
Coordinated regulation of autophagy by p38α MAPK through mAtg9 and p38IP (2009)
Figure 1

mAtg9 binds to p38IP in vitro and in vivo. (A) The AH109 strain was co‐transformed with pGBKT7 containing the C‐terminal (CT) domain of mAtg9 (bait) and pGADT7, or pACT2, containing the CT domain of p38IP (prey). The interaction was then visualized by growth of co‐transformed yeast on SD‐Trp/Leu/His plates for 3 days at 30°C. (B) Domain organization of p38IP, the N‐ (1–487 aa) and C‐ (487–733 aa) terminal domains. p38IP has a predicted nuclear localization signal (NLS), a PEST domain, and two serine‐rich domains. The region of interaction with p38α and mAtg9 is indicated. (C) HEK293A cells were transfected with RFP–mAtg9 and HA–p38IP. HA–p38IP was immunoprecipitated with an anti‐HA antibody and the co‐immunoprecipitated RFP–mAtg9 was detected with an anti‐mAtg9 antibody. HA–p38IP was detected with an anti‐HA antibody. Asterisk indicates a non‐specific band. Indirect immunofluorescence analysis of mAtg9 with (D) HA–full‐length p38IP (HA–FL), (E) HA–C‐terminal p38IP (HA–CT), and (F) HA–N‐terminal p38IP (HA–NT). Bars=5 μm. The co‐localization seen in (D) and (E) was not because of overlap of randomly distributed vesicles. Cross‐correlation function (CCF) analysis (van Steensel et al, 1996) showed a peak and maximum Rp value around ΔX=0, indicating that co‐localization between mAtg9 and p38IP is positively correlated and non random (Supplementary Figure S1).
Coordinated regulation of autophagy by p38α MAPK through mAtg9 and p38IP (2009)
Figure 2

p38IP is found on membranes, co‐localizes with mAtg9 and is required for starvation‐dependent trafficking of mAtg9. (A) HEK293A cells were transfected with HA–p38IP alone or co‐transfected with HA–p38IP and RFP–mAtg9, homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol. Equal protein amounts were resolved by SDS–PAGE and immunoblotted with anti‐mannose‐6‐phosphate receptor (MPR) antibody as a control for membrane‐associated proteins, anti‐superoxide dismutase (SOD) antibody as a control for cytosolic proteins, anti‐mAtg9 antibody, and anti‐HA antibody. HA–p38IP levels were quantified using ImageJ software and plotted relative to HA–p38IP in the PNS (n=4) (**P=0.0051). (B) HEK293 cells were depleted of mAtg9 using siRNA, then transfected with HA–p38IP and incubated in full medium or EBSS (data not shown). The asterisk indicates mAtg9‐depleted cells. Bar is 5 μM. The intensity of the nuclear versus cytosolic fluorescence in control and cells depleted of mAtg9 under fed and starvation conditions was analysed using ImageJ software (***P=0.0001, Students t‐test). (C) HEK293A cells were transfected with HA–p38IP and processed for indirect immunofluorescence. HA–p38IP was detected with an anti‐HA antibody and endogenous mAtg9 was detected with an anti‐mAtg9 antibody. HA–p38IP localizes to the nucleus and cytoplasmic puncta in full medium or EBSS. HA–p38IP co‐localizes with mAtg9 in the periphery of the cell (arrowheads). Bars (upper panels) 5 μM, (lower panels) 1 μM. (D) Anti‐mAtg9 antibody was used to immunoprecipitate mAtg9 from HEK293 cells transfected with HA–p38IP alone or with RFP–mAtg9 and HA–p38IP. A total of 5% of lysates or immunoprecipitates were probed with anti‐HA or anti‐mAtg9 antibodies. The left and right hand bottom panels were obtained from the same immunoblot, but exposed for different times to allow the endogenous mAtg9 to be visualized better. (E) HEK293 cells were transfected with control or p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium or EBSS for 2 h, then fixed and immunostained to detect endogenous mAtg9 localization. mAtg9 localization was quantified by visually scoring cells for either a juxta‐nuclear or dispersed phenotype. Bars=5 μm (data are represented as mean±s.e.m. of 200 cells, *P=0.0413, Student's t‐test).
Coordinated regulation of autophagy by p38α MAPK through mAtg9 and p38IP (2009)
Figure 3

p38IP is required for mammalian autophagy.(A) 293/GFP–LC3 cells were transfected with control or p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium (FM), full medium with leupeptin (FM Leu), EBSS (ES), or EBSS with leupeptin (ES Leu) for 2 h. FM Leu (data not shown) is identical to FM alone. Quantification of GFP–LC3‐positive autophagosome number was performed by counting in a blinded experiment. Bars=5 μm (data are represented as mean±s.e.m. of 60 cells, ***P=0.0002, Students t‐test). (B) HEK293A cells were transfected with siRNA control, siRNA for p38IP, and siRNA for ULK1. Cell lysates were analysed by SDS–PAGE for endogenous LC3 lipidation using an anti‐LC3 antibody, or immunoblotted with anti‐ULK1 antibody. (C) HeLa cells were transfected with siRNA control, and siRNA for p38IP. After incubation for 2 h as in (A), samples were immunoblotted with anti‐LC3 and anti‐actin antibodies. Endogenous LC3II/LC3I levels were quantified and the ratio presented as arbitrary units. In (B), n=4, ***P=0.0001 and *P=0.0324, Students t‐test. In (C), data are representative of two experiments. (D) 293/GFP–LC3 cells were transfected with siRNA control or siRNA p38IP, and incubated in full medium, or for 2 and 4 h in EBSS. Samples were immunoblotted with anti‐p62 antibodies, and actin as a loading control. (E) HEK293A cells were transfected with HA–p38IP or myc–ULK1 C‐terminal domain (CTD). After 24 h, cells were labelled with [14C]valine as described in Material and Methods section, and incubated in either full medium or EBSS for 2 h. Cells were then collected and analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS mock versus EBSS HA–p38IP (***P=0.036); EBSS mock versus ULK1 CTD (***P=0.0001); Students t‐test).
Coordinated regulation of autophagy by p38α MAPK through mAtg9 and p38IP (2009)
Figure 4

Activation of p38 inhibits autophagy and mAtg9 trafficking. (A) 293/GFP–LC3 cells were treated with 10 μM anisomycin for 30 min, or exposed to UV irradiation for 3 min followed by a 40‐min recovery and incubation in full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells were then fixed and visualized by confocal microscopy. GFP–LC3‐positive structures per cell were quantified as in Figure 3A. Bars=5 μm. Data are represented as mean±s.e.m. n=60 cells, mock versus anisomycin EBSS (***P=0.0001); mock versus UV EBSS (***P=0.0001); mock versus anisomycin EBSS with leupeptin (***P=0.0001); mock versus EBSS with leupeptin UV (***P=0.0001). All analysed using Student's t‐test. (B) HEK293A cells were pretreated with 10 μM anisomycin for 30 min, or UV irradiation for 3 min followed by a 40‐min recovery, before incubation in EBSS for 2 h. Images were analysed as in Figure 2E. Bars=5 μm. Data are represented as mean±s.e.m. of 60 cells, EBSS mock versus EBSS anisomycin (*P=0.0142); EBSS mock versus EBSS UV (*P=0.0150); Student's t‐test.