Lactobacillusgasseri SF1183 Affects Intestinal Epithelial
`Cell Survival and Growth
`Blanda Di Luccia1.
`, Nicola Manzo1.
`Alessandra Pollice1*
`
`, Loredana Baccigalupi1, Viola Calabro` 1, Elvira Crescenzi2, Ezio Ricca1,
`
`1 Department of Biology, University of Naples Federico II-MSA-Via Cinthia, Naples, Italy, 2 Istituto di Endocrinologia ed Oncologia Sperimentale-CNR-via S. Pansini, Naples,
`Italy
`
`Abstract
`
`It is now commonly accepted that the intestinal microbiota plays a crucial role in the gut physiology and homeostasis, and
`that both qualitative and quantitative alterations in the compositions of the gut flora exert profound effects on the host’s
`intestinal cells. In spite of this, the details of the interaction between commensal bacteria and intestinal cells are still largely
`unknown and only in few cases the molecular mechanisms have been elucidated. Here we analyze the effects of molecules
`produced and secreted by Lactobacillus gasseri SF1183 on human intestinal HCT116 cells. L. gasseri is a well known species
`of lactic acid bacteria, commonly associated to the human intestine and SF1183 is a human strain previously isolated from
`an ileal biopsy of an healthy volunteer. SF1183 produces and secretes, in a growth phase-dependent way, molecule(s) able
`to drastically interfere with HCT116 cell proliferation. Although several attempts to purify and identify the bioactive
`molecule(s) have been so far unsuccessful, a partial characterization has indicated that it is smaller than 3 kDa, thermostable
`and of proteinaceous nature. L. gasseri molecule(s) stimulate a G1-phase arrest of the cell cycle by up-regulation of
`p21WAF1 rendering cells protected from intrinsic and extrinsic apoptosis. A L. gasseri-mediated reduction of apoptosis and
`of cell proliferation could be relevant in protecting epithelial barrier integrity and helping in reconstituting tissutal
`homeostasis.
`
`Citation: Di Luccia B, Manzo N, Baccigalupi L, Calabro` V, Crescenzi E, et al. (2013) Lactobacillus gasseri SF1183 Affects Intestinal Epithelial Cell Survival and
`Growth. PLoS ONE 8(7): e69102. doi:10.1371/journal.pone.0069102
`
`Editor: Henri Salmon, INRA, UR1282, France
`
`Received March 22, 2013; Accepted June 6, 2013; Published July 23, 2013
`Copyright: ß 2013 Di Luccia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
`unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
`
`Funding: This work was supported by the FARO project (terza tornata) of the University of Naples and Istituto San Paolo. The funders had no role in study design,
`data collection and analysis, decision to publish, or preparation of the manuscript.
`
`Competing Interests: The authors have declared that no competing interests exist.
`
`* E-mail: apollice@unina.it
`. These authors contributed equally to this work.
`
`Introduction
`
`Several recent studies have shown that commensal bacteria,
`forming the human gut microbiota, establish complex symbiotic
`interactions with cells of the GastroIntestinal Tract (GIT) and that
`these interactions
`significantly contribute to human health
`[1,2,3,4]. Metagenomic experiments have indicated that the vast
`majority of the intestinal bacteria belong to two phyla, the Gram-
`negative Bacteroidetes and the Gram-positive Firmicutes, includ-
`ing the large class of Clostridia and the lactic acid bacteria [5,6].
`However, the composition of the gut microbiota is known to
`change transiently as a consequence of a variety of factors such as
`age, diet, enteral
`infections, pharmacological
`treatments and
`immunosuppression [7,8,9]. Changes in the microbiota composi-
`tion have also been associated to several diseases, such as chronic
`inflammation of the GIT, diabetes and obesity [7,10,11,12,13,14],
`and the oral administration of members of the microbiota has
`been considered as a potential clinical tool to relieve intestinal
`dysfunctions [15,16,17,18,19,20]. Interest in the beneficial func-
`tions of the human microbiota has resulted in the selection of
`specific strains with putative health-promoting capacities that are
`recognized as probiotics and are generally selected from isolates of
`the Lactobacillus or Bifidobacterium species. Probiotic bacteria have
`been shown capable to modulate systemic inflammation, cell
`
`proliferation and apoptosis, and such properties proposed as useful
`for future immunomodulatory and cancer prevention strategies
`[13,14,21,22]. In vitro studies have reported the anti-proliferative
`and pro-apoptotic effects of Lactobacillus and Bifidobacterium spp. in
`various cancer cell lines [23,24,25,26], while in vivo studies have
`shown the inhibitory activity of probiotics on liver, bladder and
`colon tumours in animal models [27,28,29,30].
`The molecular mechanisms of interaction between intestinal
`cells and bacteria have been studied in detail only in few cases and
`often quorum-sensing autoinducers, communication molecules
`released by bacteria at high densities, have been shown to
`modulate host responses either directly or through regulation of
`bacterial genes involved in gut colonization and host signaling
`[31,32]. An example in this context
`is
`the quorum-sensing
`pentapeptide CSF (Competence and Sporulation Factor) of
`Bacillus subtilis that is taken up by Caco-2 cells via the membrane
`transporter OCTN2 (organic cation transporter 2) and that
`contributes
`to eukaryotic cell homeostasis activating survival
`pathways
`(p38 MitogenActivatedProteinKinase (MAPK) and
`protein kinase B) [33]. In other cases the secreted bacterial
`effectors have not been identified: still unidentified molecules
`secreted by Lactobacillus rhamnosus GG were shown to prevent
`cytokine-induced apoptosis on two different intestinal cell model
`systems (YAMC-young adult mouse colon; HT29-colon carcino-
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`ma) [34]; molecules secreted by L. reuteri were shown to potentiate
`tumour necrosis factor (TNFa)-induced apoptosis in myeloid
`leukemia derived cells. In the latter example L. reuteri molecules
`were found to: i) suppress NF-kB activation by inhibiting IkBa
`degradation; ii) downregulate nuclear factor-kB (NF-kB)-depen-
`dent gene products affecting cell proliferation and survival; iii)
`promote apoptosis by enhancing mitogen-activated protein kinase
`(MAPK) activities including c-Jun N-terminal kinase and p38
`MAPK [35].
`Lactobacillus gasseri is a well characterized species of low GC
`gram-positive bacteria, known to represent one of
`the major
`homofermentative Lactobacillus of the human intestine [36]. We
`have isolated the SF1183 strain of L. gasseri from an ileal biopsy of
`a human healthy volunteer and, in particular, from the fraction of
`bacteria tightly associated to the epithelial cells. SF1183 was
`shown to have antimicrobial activity against a panel of entero-
`pathogens and to form a matrix (biofilm) in standard laboratory as
`well as in simulated intestinal conditions [36].
`This study investigates the effects of molecules produced and
`secreted by L. gasseri SF1183 on colorectal HCT116 cells, both at
`the molecular and cellular
`level. Since HCT116 cells are
`responsive to TNFa-induced apoptosis [37,38], we tested their
`response to the presence of L.
`gasseri SF1183 supernatant.
`Moreover, we extended our analysis to the effects of another
`inducer of apoptosis to evaluate the specificity of the observed
`effect.
`
`Results and Discussion
`
`The Conditioned Medium (CM) of L. gasseri SF1183
`Protects HCT116 cells from TNFa Induced Apoptosis
`Among the most common features of chronic intestinal
`inflammations, such as Crohn and irritable bowel diseases (IBDs),
`is the increase in the production of
`inflammatory cytokines,
`epithelial cell apoptosis and immune cell infiltration, leading to
`disruption of the intestinal epithelial integrity. TNFa is among the
`cytokines more largely produced under these conditions. It is
`known to regulate both anti- and pro-apoptotic signaling pathways
`and determine the cell fate by controlling the balance between the
`two pathways [39]. To study the effects of molecules secreted by L.
`gasseri on TNFa-induced apoptosis we used the TNFa sensitive
`HCT116 human colon cancer cells as a model of
`intestinal
`epithelial cells [37]. As a marker of apoptosis we followed the
`proteolytic cleavage of PARP-1, a regulator of the DNA base
`excision repair pathway essential for the maintenance of genomic
`integrity and for survival in response to genotoxic insults [40].
`PARP-1 is known to be specifically proteolysed by the Caspase 3
`to a 24 kDa DNA-binding domain (DBD) and a 89 kDa catalytic
`fragment during the execution of the apoptotic program [41]. To
`set up the experimental conditions, HCT116 cells were incubated
`with 1 nM TNFa for various times and cell extracts analyzed by
`western blotting with anti-PARP-1 antibody. As
`shown in
`Figure 1A, the amount of proteolyzed PARP-1 increased with
`the time of exposure to TNFa. Therefore we decided to use
`8 hours of treatment with 1 nM TNFa to detect either induction
`or inhibition of PARP cleavage,
`for all
`therein experiments
`involving a TNF-a activation.
`A filter-sterilized conditioned medium (CM) of a L. gasseri
`SF1183 culture was added (20% v/v)
`to HCT116 cells and
`incubated for 16 hours. Then, TNFa was added and, after
`additional 8 hours of incubation, cells were harvested and whole
`extracts analyzed by western blotting with anti-PARP-1 antibody.
`As shown in Figure 1B the bacterial CM alone did not have any
`
`Effects of Probiotic L.gasser on Intestinal Cells
`
`effect on PARP-1 cleavage while was able to significantly reduce
`the TNFa-induced proteolytic activation of PARP-1.
`L. gasseri is a homofermentative bacterium that, therefore, grows
`producing lactic acid as
`the only metabolic end-point of
`carbohydrate metabolism. As a consequence, its growth medium
`is acidified during growth to reach a final pH value of 4.0. To
`verify that the reduction in the extent of PARP-1 cleavage was due
`to secreted molecules and not to the acidification of the growth
`medium, the same experiment of Figure 1B was performed adding
`to HCT116 cells the same amount (20% v/v) of the fresh bacterial
`growth medium (MDM) either at its normal pH (pH 7.0) or
`acidified to pH 4.0 with lactic acid. As shown in Fig. 1C, both
`media did not have any effect on TNFa-induced cleavage of
`PARP-1 suggesting that the CM of L. gasseri SF1183 contains
`molecules with anti-apoptotic activity.
`
`The CM of L. gasseri SF1183 Contains Bioactive Soluble
`Molecule(s) Secreted During the Stationary Phase of
`Growth
`step toward the characterization of molecule(s)
`As a first
`involved in the observed effect, we decided to size-separate the
`CM of L. gasseri by using a 3 kDa molecular mass cut-off filter. As
`Figure 2A clearly shows we observed bioactivity largely in the
`filtrate, indicating a small (less than 3 kDa) molecular mass for the
`effector(s) molecule(s). Further, different enzymatic treatments of
`the CM indicated that bioactivity is proteinase-K sensitive,
`suggesting a proteinaceous nature (Figure 2B). After a heat
`treatment of 30 minutes at 100uC the CM was still able to reduce
`the TNFa-induced cleavage of PARP-1 at the same extent of
`untreated CM (Figure 2C),
`suggesting that
`the bioactive
`molecule(s) is not thermolabile. Often bacteria secrete bioactive
`molecules during their stationary phase of growth. We thus tested
`the CM of L. gasseri cultures at different stages of growth and
`observed bioactivity produced only in early and late stationary
`phase of growth (24 and 48 hours of growth, respectively)
`(Figure 2D). All experiments
`therein reported have been
`performed by using the size-fractionated (,3 kDa) CM of a late
`stationary culture of L. gasseri SF1183.
`
`The CM of L. gasseri SF1183 Affects Cell Proliferation of
`HCT116 Cells
`To characterize the cellular response to L. gasseri secreted
`molecules, we analyzed HCT116 cell number and viability after
`growth in presence of CM. Briefly, cells were incubated for 24
`hours with CM of L. gasseri (20% vol/vol) and then analyzed both
`for the number of cells by counting in a Burker chamber and for
`cell viability by MTS assay. As Figure 3A shows, the CM caused a
`30% reduction in the number of cells. The MTS assay (Figure 3B)
`showed a reduction in cell viability of
`the same order of
`magnitude.
`To get more insights into the cellular response, we looked at the
`cell-cycle distribution profile and at the expression of cell cycle-
`related molecular markers in HCT116 cells exposed to TNFa
`and/or to the CM of L. gasseri.
`The cell cycle distribution was analyzed by flow cytometry and
`showed that treatment with TNFa causes a drastic increase in the
`subG1 cell population (from 4 to 28%) while the pre-treatment of
`cells with the CM of L. gasseri strongly reduced the TNFa induced
`effect (Figure 4A), thus supporting our previous data indicating a
`reduction in the extent of PARP-1 cleavage (see Figure 1B,
`2B, 2C). Importantly, we found that the CM alone caused a
`significant increase (up to 18%) in the G1 population of cells with a
`compensatory decrease in S/G2 cells, indicating that cells were
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`Effects of Probiotic L.gasser on Intestinal Cells
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`Figure 1. HCT116 cell response to L.gasseri CM with or without TNFa treatment. Western blot with anti-PARP-1 antibody of whole cell
`extracts from HCT116 cells incubated in (A) complete cell culture medium supplemented or not with TNFa (1 nM) for 2, 8 or 24 hours; (B) complete
`cell culture medium supplemented or not with CM (20%v/v) for 16 hours before treatment with 1 nM TNFa for 8 hours; (C) complete cell culture
`medium supplemented or not with TNFa (1 nM) for 8 hours and MDM (20%v/v) or MDM+lactic acid pH4 (20%v/v). After the treatments cells were
`collected, lysed and protein concentration determined. Equal amount of cell lysates were fractionated on SDS-PAGE and analyzed by western blotting
`with antibodies against PARP-1. Actin was used as a loading control.
`doi:10.1371/journal.pone.0069102.g001
`
`unable to resume the cell cycle at normal phase transit rate
`(Figure 4A), consistently with previous MTS and proliferation
`data. This suggests that, indeed, molecules secreted from L. gasseri
`can drastically interfere with proliferation of HCT116 cells,
`rendering them less prone to TNFa induced apoptosis.
`Expression of the cell cycle markers p21WAF1 and pERKs was
`also investigated to explore the effects of the CM of L. gasseri at the
`molecular level. p21WAF1 (also known as cyclin-dependent kinase
`inhibitor 1) is a regulator of cell cycle progression at the S phase
`that acts as an inhibitor of cyclin-dependent kinase, and occupies a
`central position in the regulation of the cell cycle in many tissues
`[42,43]. Levels of p21WAF1 protein are regulated during the cell
`cycle at
`the levels of
`transcription and protein degradation,
`although many questions remain on the mechanism of p21
`proteolysis
`[44,45]. Extracellular
`signal-regulated
`kinases
`(ERK1,2) are members of the MAPK super family that can
`mediate cell proliferation and apoptosis. Activated (phosphorylat-
`ed) ERKs, are usually associated with active cell proliferation [46],
`while p21 increase correlates with a G1 cell cycle arrest [47].
`Immunoblots with the appropriate antibodies
`showed that
`treatment with CM significantly induced p21WAF independently
`from TNFa (Figure 4B; compare lanes 1–2 with 3–4) while pERKs
`expression was inhibited in CM treated cells, strongly supporting
`
`the antiproliferative effect of molecule(s) present
`supernatant.
`Altogether these experiments clearly indicate that L. gasseri
`supernatant exerts a cytostatic but not a cytotoxic effect on
`epithelial colon cells.
`
`in L. gasseri
`
`The CM of L. gasseri SF1183 Protects HCT116 Cells from
`Cisplatin Induced Apoptosis
`in L. gasseri
`To test whether bioactive molecules present
`supernatant could exert anti-apoptotic effects against other
`apoptosis-inducers we preincubated HCT116 cells with CM and
`then treated them with 30 mM cisplatin to induce the intrinsic
`apoptotic pathway. As
`shown in Figure 5A cytofluorimetric
`analysis indicate that a G1 cell cycle arrest is induced by CM
`addition which causes cells to be more resistant
`to cisplatin
`induced apoptosis. These observations are supported, at
`the
`molecular level, with an increase in p21WAF1 levels and a
`decrease of ERKs activation when CM was added to the cells
`(Figure 5B, lanes 1,2). Consistently, pretreatment of cells with CM
`determined a reduction in the extent of PARP-1 cleavage when
`cells were subjected to cisplatin action (Figure 5B, lanes 3,4).
`Altogether our results clearly indicate that probiotic L. gasseri
`protects
`intestinal epithelial cells
`from apoptosis
`induced by
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`Effects of Probiotic L.gasser on Intestinal Cells
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`Figure 2. L.gasseri secretes thermostable, bioactive molecule(s) of proteinaceous nature during the stationary phase of growth.
`HCT116 cells were incubated in complete cell culture medium supplemented or not with TNFa (1 nM) for 8 hours and with A) CM fractionated with a
`cut-off of 3 kDa, or B) CM treated with different enzymes [Trypsin, Proteinase K, DNAse I, RNAse A], or C) CM treated at 100uC for 30 minutes, or D) CM
`of cultures at the indicated phases of growth. After the treatments, cells were collected, lysed and total cell extracts were analyzed by western
`blotting with antibodies against PARP-1. Actin was used as a loading control. PARP-1 band intensity was evaluated by ImageQuant analysis on at least
`two different expositions to assure the linearity of each acquisition. Values expressed as ratio with the corresponding actin values and normalised to
`the reference point (PARP-1 cleavage in medium). Percentage of increase (+) or decrease (–) with respect to the intensity of the reference point are
`indicated.
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`Effects of Probiotic L.gasser on Intestinal Cells
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`bacterial host responses [31,32,33]. Unfortunately, the definition
`of the chemical nature of the molecule(s) secreted by L. gasseri
`SF1183 and able to affect HCT116 cells has been so far
`unsuccessful. The size-fractionated (less than 3 kDa) CM of L.
`gasseri was analyzed by gel
`filtration chromatography with a
`Superdex Peptide 10/300 GL (GE Healthcare Life Sciences)
`column and two main peaks were obtained (Fig. S1 in File S1).
`Chromatographic fractions containing either one of the two peaks
`were tested for the ability to reduce the TNFa-induced cleavage of
`PARP-1 (Fig. S2A in File S1). Only one of the fractions (Fraction
`1) was shown to reduce the TNFa-induced cleavage of PARP-1 at
`the same extent of the unfractionated CM (Fig. S2B in File S1).
`Unfortunately, attempts to analyze Fraction 1 by mass-spectrom-
`etry have been so far unsuccessful, probably because of
`the
`minimal concentration of molecules in the fraction. To define the
`chemical nature of the molecule(s) affecting HCT116 cells and
`identify its cellular and molecular targets will then be a future and
`challenging task.
`
`Materials and Methods
`
`Bacterial Growth and Preparation of Conditioned
`Medium
`Lactobacillus gasseri (SF1183) was grown in MRS broth (Difco,
`Detroit, MI) for 24 hours at 37uC and the culture diluted and used
`to inoculate MDM (Glucose 10 g/L, Sodium acetate 5 g/L,
`KH2PO4 3 g/L, K2HPO4 3 g/L, MgSO4 *7H2O 0.2 g/L, L-
`Alanine 100 mg/L, L-Arginine 100 mg/L, L-Aspartic acid
`200 mg/L, L-Cysteine 200 mg/L, L-Glutamic 200 mg/L, L-
`Histidine 100 mg/L, L-Isoleucine 100 mg/L, L-Leucine 100 mg/
`L, L-Lysine 100 mg/L, L-Methionine 100 mg/L, L-Phenylala-
`nine 100 mg/L, L-Serine 100 mg/L, L-Tryptophan 100 mg/L,
`L-Tyrosine 100 mg/L, L-Valine 100 mg/L, Nicotinic acid 1 mg/
`L, Pantothenic acid 1 mg/L, Pyridoxal 2 mg/L, Riboflavin 1 mg/
`L, Cyanocobalamin 1 mg/L, Adenine 10 mg/L, Guanine 10 mg/
`L, Uracil 10 mg/L) minimal medium. Cells of SF1183 were then
`grown anaerobically for 48 hours at 37uC. The culture was
`centrifuged (1000 g for 10 min at RT) and the supernatant
`(conditioned medium, CM) was
`filtered-sterilized through a
`0.22 mm low-protein binding filter (Millipore, Bedford, MA,
`US). CM treated with proteases and nucleases was prepared as
`described above and size fractionated (3-kDa cutoff spin column;
`Centricon, Millipore). Before treatment with trypsin (GIBCO) or
`proteinase K (Invitrogen), or DNasi I, or RNasi A (Invitrogen, Life
`Technology, Monza, Italy) at a final 100 mg/ml concentration for
`60 min at 37uC the pH of CM was neutralized with concentrated
`NaOH (10 N). After the enzymatic treatments CM was acidified to
`pH 4.0 using concentrated HCl and fractionated as described
`above to remove the enzymes.
`
`Cell Culture and Treatment with Bacterial CM
`HCT116 cells (ATCC CCL 247) derived from a poorly-
`differentiated colonic adenocarcinoma and were maintained in
`RPMI 1640 supplemented with 10% fetal bovine serum and 1%
`penicillin-streptomycin. Cells were cultured at 37uC in humidified
`atmosphere of 5% CO2. The bacterial CM was employed for the
`treatment at 20% v/v concentration in complete growth medium.
`After incubation of 16 hours with CM (20% v/v), TNFa (1 nM)
`(Millipore, Milan, Italy) or Cisplatin (30 mM) (Sigma Milan, Italy)
`was added and cells harvested after 8 hours or 24 hours of
`treatment. Cells were lysed and cell extracts prepared for Western
`blot and FACS analysis, respectively, as described below.
`
`Figure 3. The CM of L. gasseri SF1183 affects HCT116 cell
`number but not cell viability. Proliferating HCT116 cells were
`incubated in complete cell culture medium supplemented or not with
`CM (20%v/v). After 24 hours (A) controls (NT) and CM-treated (CM) cells
`were collected and counted in a Burker chamber; or (B) incubated with
`3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-
`nyl)-2H-tetrazolium as a substrate and the absorbance of converted
`formazan measured at 490 nm.
`doi:10.1371/journal.pone.0069102.g003
`
`inflammatory cytokines or cytotoxic drugs, causing cell cycle
`arrest.
`
`Conclusions
`The main result of this report is that the conditioned medium of
`a stationary culture of the human isolate SF1183 of L. gasseri
`contains molecule(s) able to affect cell proliferation of HCT116
`cells, protecting them from intrinsic as well as extrinsic, TNFa-
`induced, apoptosis. Chronic inflammations cause an increase in
`inflammatory cytokines (such as TNFa), epithelial cell apoptosis
`and immune cell infiltration, leading to disruption of the intestinal
`epithelial
`integrity. Therefore, a reduction of cell proliferation
`could protect epithelial barrier integrity and help in reconstituting
`tissutal homeostasis.
`The L. gasseri molecule(s) responsible of the observed effects is
`proteinaceous, has a small (less than 3 kDa) size and its synthesis is
`growth phase-dependent, occuring only in bacterial cells
`in
`stationary phase. Those properties are suggestive of bacterial
`quorum-sensing autoinducers, communication molecules pro-
`duced at high cell density and known to act as modulator of
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`Effects of Probiotic L.gasser on Intestinal Cells
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`Figure 4. The CM of L.gasseriSF1183 affects cell proliferation of HCT116 cells. Proliferating HCT116 cells were incubated in complete cell
`culture medium supplemented or not with CM (20%v/v) and/or TNFa (1 nM). After the treatments, cells were collected and treated for flow-
`cytometric analysis (A and Fig. S3 in File S1) or western blot (B) with the indicated antibodies.
`doi:10.1371/journal.pone.0069102.g004
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`SDS-PAGE and Western Immunoblot Analysis
`Cells were harvested in lysis buffer (50 mM Tris-HCl pH 7.5,
`5 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM phenylmethyl-
`sulfonyl
`fluoride, 0.5% sodium deoxycholate, and protease
`inhibitors) and total protein extract prepared as previously
`described [48]. Briefly, cell lysates were incubated on ice for 40
`minutes, and the extracts were centrifuged at 15000 g for 15
`minutes
`to remove cell debris. Protein concentration was
`determined by the Bio-Rad protein assay (Bio-Rad). After the
`addition of 2x Laemmli buffer (SIGMA), samples were boiled at
`100uC for 5 minutes and resolved by SDS-polyacrylamide gel
`electrophoresis
`(10% or 12%). Proteins were transferred to
`polyvinylidenedifluoride (PVDF) membranes (Millipore Milan,
`Italy) as previously described [49]. The membranes were blocked
`in 5% w/v milk buffer (5% w/v non-fat dried milk, 50 mM Tris,
`200 mM NaCl, 0,2% Tween 20) and incubated with primary
`antibody diluted in 5% w/v milk or bovine serum albumine buffer
`for 2 hours at room temperature or overnight at 4uC. Primary
`antibodies were anti-rabbit PARP-1 (Cell Signaling, EuroClone,
`Milan, Italy), anti-rabbit pErks 42/44 (Cell Signaling, EuroClone,
`Milan, Italy), anti-rabbit p21WAF1 (Santa-Cruz Biotechnology,
`DBA Milan-Italy), anti-goat b-actin (Santa-Cruz Biotechnology
`DBA Milan, Italy). Data were visualized by enhanced chemilu-
`minescence method (ECL, GE-Healthcare Milan, Italy) using
`
`HRP-conjugated secondary antibody (Santa-Cruz Biotechnology
`DBA Milan, Italy) incubated 1 hour at room temperature, and
`analysed by Quantity One Hsoftware of ChemiDoc TMXRS
`system (Bio-Rad Milan, Italy).
`
`Cell Growth and Flow Cytometry Analysis
`HCT116 cells were plated in 35 mm dishes at the cell density of
`2,56105 cells/plate. For cell growth analysis, cells were cultured in
`complete growth medium supplemented or not with bacterial CM
`at 20% v/v concentration for 24 hours. After the treatment, cells
`were collected and counted in a Burker chamber. Flow cytometry
`analysis was performed as previously described [50]. Briefly, cells
`were washed twice with PBS and harvested at 1500 g with 0.05%
`trypsin in 0.15% Na2EDTA. Cells were then centrifuged, washed
`in PBS, fixed with ice-cold 70% ethanol, and stored overnight at
`4uC. Fixed cells were washed in PBS and then incubated with
`propidium iodide (50 mg/ml) and RNAse A (10 mg/ml) for 30 min
`at room temperature. Data acquisition was performed using a
`CyAn ADP Flow Cytometer (Beckman Coulter, Inc., Milano,
`Italy) and Summit Software.
`
`MTS Assay
`HCT116 cells were cultured at a density of 2,56105 cells per
`well in flat bottomed 6-well plates and supplemented or not with
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`Effects of Probiotic L.gasser on Intestinal Cells
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`Figure 5. The anti-apoptotic effect of L.gasseriis not specific for TNFa-induced apoptosis. Proliferating HCT116 cells were incubated in
`complete cell culture medium supplemented or not with CM (20%v/v) and/or cisplatin (30 mM). After the treatments, cells were collected and treated
`for flow-cytometric analysis (A and Fig. S4 in File S1) or western blot (B) with the indicated antibodies.
`doi:10.1371/journal.pone.0069102.g005
`
`for 24 hours. After treatment, CellTiter 96H
`CM (20% v/v)
`AQUEOUS One Solution Reagent (Promega, Madison, WI, US)
`was added to each well according to the manufacturer’s
`instructions. After 30 minutes cell viability was determined by
`measuring the absorbance at 490 nm using a Multiscan spectrum
`(Thermo Electron Corporation).
`
`Supporting Information
`
`File S1 Supplemental figures. Figure S1, the CM of L. gasseri
`was size fractionated with a 3 kDa molecular mass cut-off filter and
`loaded on a gel
`filtration chromatographic column (Superdex
`Peptide 10/300 GL, GE Healthecare Life Sciences). The elution
`buffer was AMAC 0.3 M. Two main peaks were observed at
`220 nm. Figure S2, chromatographic fractions from the experi-
`ment of Fig. S1 were tested by western blotting with anti-PARP-1
`antibody (A). As a control, cells were also treated with the elution
`buffer (AMAC 0,3 M). (B) Densitometric analysis of the western
`blot. PARP-1 band intensity was evaluated by ImageQuant
`analysis on at least two different expositions to assure the linearity
`
`of each acquisition. Values are expressed as ratio with the
`corresponding actin values and normalised to the reference point
`increase (+) or
`(PARP-1 cleavage in medium). Percentage of
`decrease (–) with respect to the intensity of the reference point are
`indicated. Figure S3, enlargment of part of Fig. 4 showing the
`output of the FACS analysis. Figure S4, enlargment of part of
`Fig. 5 showing the output of the FACS analysis.
`(PDF)
`
`Acknowledgments
`
`We thank Elio Pizzo for helping us with the chromatography experiments
`and Luciano Di Iorio for technical assistance.
`
`Author Contributions
`
`Conceived and designed the experiments: AP ER BDL NM. Performed the
`experiments: BDL NM EC. Analyzed the data: BDL NM LB EC VC ER
`AP. Contributed reagents/materials/analysis tools: LB VC ER AP. Wrote
`the paper: ER AP.
`
`PLOS ONE | www.plosone.org
`
`7
`
`July 2013 | Volume 8 |
`
`Issue 7 | e69102
`
`Genome Ex. 1049
`Page 7 of 8
`
`

`

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