Felipe Cabezas Muñoz

<p>(A) Pax7 localization is not affected by S201/S205 phosphorylation. Immunofluorescence analysis of C3H10T1/2 cells transfected with myc-Pax7-WT, myc-Pax7-AS, myc-Pax7-SA or myc-Pax7-AA shows nuclear localization (DAPI) of all Pax7 variants (myc). Scale bar = 10μm. (B) the ability of each Pax7 mutant to repress MyoD-dependent myogenic conversion of C3H10T1/2 cells, was evaluated by immunofluorescence. All Pax7 mutants repressed myotube formation resembling the effect of Pax7-WT. Panel depicts MyoD and myc-Pax7 expression in transfected mRFP(+) cells. Quantification of three representative experiments, evaluating myotube formation by fusion index (i.e. myotube associated nuclei/ total MyoD+ or myc+ nuclei; considering ≥3 nuclei in mRFP+ cells as a myotube) is shown. Lower panel: the percentage of MyHC associated MyoD(+) nuclei was also inhibited upon Pax7 and Pax7 phospho-mutants co-expression. Bar = 10μm. (C) Transcriptional activity of Pax7-WT or Pax7 phospho-mutants, was evaluated using the <i>6xPRS9-luc</i> reporter gene in C3H10T1/2 cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154919#pone.0154919.ref020" target="_blank">20</a>]. CMV-LacZ expression vector was co-transfected and β-galactosidase activity was used to normalize luciferase activity. The graph represents the relative luciferase activity (RLA) (luciferase/β-galactosidase) / Pax7-WT activity ratio; mean±SEM, n = 3. No significant differences between Pax7-WT and mutants activity was observed. (D) Phosphorylation status of S201/S205 affects Pax7 protein levels. Upper panels: Western Blot analysis of myc-tagged Pax7 point mutants expression in C3H10T1/2 cells. GFP was used as transfection/loading control. Lower panels show quantification of myc-Pax7/GFP ratio normalized to Pax7-WT; mean±SEM, n = 3 (left), n = 4 (right); ANOVA, * p<0.05. Pax7-AA protein levels are significantly lower than Pax7-WT. Pax7-DS and Pax7-DD phospho-mimetic mutants exhibit higher expression levels compared to Pax7-WT.</p

<p>(A) TBB-induced Pax7 decline is prevented by concomitant proteasome inhibition. Proliferating C2C12 cells were treated as indicated with TBB 125 μM and/or 1 μM of the proteasome inhibitor epoxomicin (Epo) for 6 hours and analyzed by Western blotting. GAPDH was used as loading control and anti-phospho-CK2 substrate antibody was used as a control of TBB treatment. Right panel shows quantification of protein levels (Pax7/GAPDH) normalized to control (DMSO); mean±SEM, n = 3; ANOVA, * p<0.05. (B) TBB increases Pax7 ubiquitination in proliferating myoblasts. C2C12 cells were transfected with Pax7 and myc-6xHis-ubiquitin, treated with DMSO or 100 μM TBB for 12 hours and 12.5 μM MG132 was added for the last 6 hours before cell lysis. Denaturing Ni-NTA pull-down, followed by Western Blot shows higher levels of ubiquitinated Pax7 in TBB treated cells. Inputs = 10% of cell extracts prior to Ni-NTA pull-down. (C) Disruption of Pax7 phosphorylation by CK2, increases its ubiquitination <i>in vivo</i>. C2C12 myoblasts were transfected with the specified constructs for bimolecular fluorescence complementation (BiFC) and treated with 25 μM MG132 and 125 μM TBB or DMSO as indicated, for 6 hours before fixation. Interaction of bFos-VC and bJun-VN was included as a positive control for BiFC. Transfection of VC and VN was used as a negative control, exhibiting non-specific diffuse fluorescence signal. Arrows indicate positive BiFC and arrowheads shows transfected cells (mRFP positive cells) without complementation. Quantification of BiFC positive cells from total mRFP positive population mean±SEM, n = 3; ANOVA, * p<0.05.</p

<p>LLC-PK1 cells and dGBECs were treated with 2.5 or 10 ng/ml TGF-ß1 1, respectively, for 48 h, and total cell lysates (40 μg) from LLC-PK1 cells (A) and dGBECs (C) were subjected to SDS-PAGE and immunoblotted with antisera against megalin (top panel) or with E-cadherin antibodies (middle panel). The graphs below the immunoblots show the densitometric analyses of megalin and E-cadherin levels normalized with the housekeeping gene ß-tubulin (bottom panel). The results are expressed as the mean ± standard deviation (SD). Significant differences are indicated by *P≤0.05. Scale bars, 20 μm. (n = 3 independent experiments). (B and D) Immunofluorescence for megalin and E-cadherin in confluent LLC-PK1 cells and dGBEC treated with TGF-ß1 for 48 h in the presence or absence of the inhibitor SB-431542 (10 μM). Cells were grown on coverslips, fixed in 4% paraformaldehyde and double-stained using antibodies specific for megalin (B and D; green) and E-cadherin (B and D; red). Overlapping images with Hoechst-stained nuclei (blue) are shown.</p

<p>LLC-PK1 cells were treated with albumin concentrations ranging from 0.01 to 20 mg/ml for 24 h. Megalin (A) and PAI-1 mRNA levels (B) were measured by qPCR. (C) Megalin and (D) PAI-1 mRNA levels in LLC-PK1 cells that were exposed to 20 mg/ml of albumin and/or the inhibitor SB for 20 h. The results are expressed as the average RQ ± standard deviation (SD). (E) LLC-PK1 cells were transfected with the megalin promoter construct (1500PromBasic) and a ß-galactosidase vector (pCMVß) and incubated in the presence or absence of 20 mg/ml of albumin for 24h. Later, the cells were lysed, and luciferase activity was measured. (F) LLC-PK1 cells were transfected with the 1500PromBasic plasmid and SMAD2/SMAD3- siRNAs, after 24h the cells were treated with 20 mg/ml of albumin for another 24h. Luciferase activity was determined using a Luciferase Reporter Assay System, and the ß-galactosidase signal was used to normalize the results. Data are expressed as the means ± SD. Significant differences with controls are indicated by: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Significant differences between treatments are indicated by # P≤0.05, ## P ≤ 0.01.</p

<p>LLC-PK1 cells were treated with DAPT 10 μM or vehicle for 24 h. During the last 10 h, cells we treated with 2.5 or 20 ng/ml TGF-ß1. (A, C) Cell lysates were processed by SDS-PAGE and probed with anti-megalin or anti-tubulin antibodies. (B, D) The graphs show the densitometric analysis of Megalin CTF/tubulin ratio and of full length Megalin/tubulin ratio with and without DAPT. The results are expressed as the mean ± SEM (n = 4).</p

<p>(A) Schematic representation of SBEs and site-directed mutations (asterisks) generated in the human megalin promoter constructs. (B) LLC-PK1 cells were transfected with wild type or mutant SBE constructs of the human megalin promoter (1500PromBasic) and with the ß-galactosidase vector (pCMVß). Co-transfections were performed in the presence (black bars) or absence (white bars) of SMAD2 and SMAD3 cDNAs. After 24 h, the transfected cells were lysed, and luciferase activity was measured. Luciferase activity was normalized with ß-galactosidase expression to determine transfection efficiency. The results are expressed as the means ± SD. Significant differences with the respective controls (untransfected SMAD factors) are indicated by *P≤ 0.001. Significant differences with the wild type construct are indicated by #P≤0.001. (C) LLC-PK1 cells expressing wild type or mutant SBE constructs of the human megalin promoter (1500PromBasic) and ß-galactosidase vector (pCMVß) were incubated in the presence or absence of 2.5 ng/ml of TGF-ß1 for 24h. Later, the cells were lysed, and luciferase activity was measured. The results are expressed as the means ± SD. Significant differences with the respective controls are indicated by ∞P≤ 0.05. Significant differences with the wild type construct are indicated by &&P≤0.01, &&&P≤0.001.</p

<p>24 h after isolation, mouse primary myoblasts were incubated with 100 μM TBB or DMSO for 6 hours before fixation. Indirect immunofluorescence for Pax7, Syndecan-4 (A, B), MyoD (A), Myogenin (B) was performed as indicated. TBB treatment results in a significant increase in the percentage of cells expressing reduced Pax7 levels, while expressing high levels of myogenin (arrows). (C) Quantification of cell subpopulations present in (B), shows a ~10-fold increase in the percentage of Pax7(-)/ myogenin (+) cells (i.e. differentiating cells) with a concomitant decrease in the percentage of proliferating Pax7(+)/myogenin(-) sub population. mean±SEM, n = 3. (D) qPCR analysis determining relative Pax7 mRNA expression upon CK2 inhibition (sample pool obtained from one experiment performed as in A and B).</p

<p>(A) LLC-PK1 cells were seeded in triplicate in 24-well plates. The cells were transfected 48 h later with the megalin promoter construct (-1500PromBasic) and a ß-galactosidase vector (pCMVß). Co-transfections were performed in the presence or absence of plasmids encoding for SMAD2 and SMAD3. After 24 h, the transfected cells were lysed, and luciferase activity was measured. Cells expressing the megalin promoter construct showed a 50% decreased in luciferase activity when the SMAD transcription factors were overexpressed. (B) The overexpression of SMAD factors increased luciferase expression from the p800luc plasmid (positive control). Data in (A) and (B) are expressed as the means ± SD, and significant differences with the control (pCMV5) are indicated by *P≤ 0.001. (C) LLC-PK1 cells were transfected with the megalin promoter construct (1500PromBasic) and a ß-galactosidase vector (pCMVß). After 24h the cells were incubated in the presence or absence of TGF-ß1 2.5 ng/ml for another 24h and luciferase activity was measured in the cell lysates. Cells expressing the megalin promoter construct in the presence of TGF-ß1 show decreased luciferase activity. (D) Positive control with the p800luc plasmid in which TGF-ß1 increases luciferase expression. Data in (C) and (D) are expressed as the means ± SD, and significant differences with the control are indicated by #P≤0.05. (E) LLC-PK1 cells were transfected with pCMV5 or with SMAD2 and SMAD3 cDNAs. After 48 h of expression, the cells were lysed and megalin, SMAD2 and SMAD3 were analyzed by immunoblot; tubulin was used as a loading control. (F) Quantification of megalin/tubulin ratio in cells transfected with SMAD2 and SMAD3. (G) LLC-PK1 cells, expressing pCMV5 or SMAD2 and SMAD3 cDNAs were permeabilized with 0.1% saponin and incubated with anti-megalin and anti-FLAG. Cells were then incubated with anti-mouse Alexa-647 and anti-rabbit Alexa -488 antibodies and analyzed by flow cytometry. The results are plotted as the % of the total megalin observed in SMAD2/3 positives cells. Data in (F) and (G) are expressed as the means ± SE, and significant differences with the control are indicated by #P≤0.05.</p

<p><b>(</b>A) LLC-PK1 cells were transfected with control siRNA o SMAD2/SMAD3-specific siRNAs. After 48 h of expression, SMAD2 and SMAD3 were analyzed by immunoblot and tubulin was measured as a loading control (B) Quantification of SMAD2/tubulin or SMAD3/tubulin in cells transfected with siRNA. (C, D) SMAD2/SMAD3 were silenced with SMAD2/SMAD3- siRNAs in LLC-PK1 cells. The cells were then transfected with the megalin promoter construct 1500PromBasic (C) or the p800luc plasmid (D). After 24h the cells were incubated with vehicle or 2.5 ng/ml of TGF-ß1for another 24h. Luciferase activity was determined using a Luciferase Reporter Assay System, and the ß-galactosidase signal was used to normalize the results. Data are expressed as the means ± SD, and significant differences with the control are indicated by *P≤0.05, while significant differences between non silenced and silenced cells are indicated by ### P≤0,001. (E, F) LLC-PK1 cells were transfected with control siRNA o SMAD2/SMAD3-siRNAs. After 24 h of expression, cells were cultured in low serum medium (0.5% FBS) for 16 h and then treated in the presence or absence of 2.5 ng/ml of TGF-ß1 or 24h. After 60h of silencing and 24h of treatment with TGF-ß1, the cells were lysed and the analyzed by immunoblot. In (E) the expression of megalin, SMAD2 and SMAD3 were determined and the detection of tubulin was used as a loading control. (F) Shows the quantification of megalin/tubulin, as % of the control, in cells transfected with siRNA and treated with TGF-ß1. Data are expressed as the means ± SE. Significant differences with the control are indicated by *P≤0.05.</p

<p>(A) LLC-PK1 cells and (C) dGBECs were treated with the indicated TGF-ß1 concentrations for 12 h, and megalin mRNA levels were analysed by real-time PCR. Megalin mRNA levels analysed by real-time PCR after treating LLC-PK1 cells (B) and (D) dGBECs with 2.5 or 10 ng/ml TGF-ß1 respectively for different times (6, 12, 30 and 48 h). As control, we measured PAI-1 mRNA levels in LLC-PK1 cells (E) that had been treated with TGF-ß1 at the indicated concentrations for 12 h or (F) treated with 2.5 ng/ml TGF-ß1 for different times (as shown in B and D). Values were normalized using either actin or GADPH as a housekeeping gene (n = 3 independent experiments). The results are expressed as the means ± standard deviation (SD). Significant differences compared with the control (vehicle) are indicated by *P≤0.05, **P≤0.01, ***P≤0.001.</p

The biological properties of chilean propolis have been described and include antibacterial, antifungal and antibiofilm activities. Propolis has a strong antimicrobial potential. Clinical experiences with synthetic antibiotics indicated the need to discover new sources of bioactive compounds associated with ethnopharmacological knowledge or natural sources such as propolis. The microscopic analysis of pollen grains from plants allows us to determine the botanical origin of the propolis samples. In Angol, sample pollen grains were obtained from fodder plants (Sorghum bicolor; Lotus sp.) and trees, such as Acacia sp., Pinus radiata, Eucalyptus sp. and Salix babylonica. Propolis from the Maule region contains pollen grains from endemic plants such as Quillaja saponaria. Finally, the sample obtained from Melipilla presented a wider variety of pollen extracted from vegetable species.Colorimetric assays performed to quantify the total polyphenols present in Chilean propolis samples establ...

Satellite cells (SCs) are myogenic progenitors responsible for skeletal muscle regeneration and maintenance. Upon activation, SCs enter a phase of robust proliferation followed by terminal differentiation. Underlying this myogenic progression, the sequential expression of muscle regulatory transcription factors (MRFs) and the downregulation of transcription factor paired box gene 7 (Pax7) are key steps regulating SC fate. In addition to transcriptional regulation, post‐translational control of Pax7 and the MRFs provides another layer of spatiotemporal control to the myogenic process. In this context, previous work showed that Pax7 is ubiquitinated by the E3 ligase neural precursor cell‐expressed developmentally downregulated protein 4 and interacts with several proteins related to the ubiquitin–proteasome system, including the deubiquitinase ubiquitin‐specific protease 7 (USP7). Although USP7 functions in diverse cellular contexts, its role(s) during myogenesis remains poorly explor...

Megalin/LRP2 is a receptor that plays important roles in the physiology of several organs, such as kidney, lung, intestine, and gallbladder; and also in the physiology of the nervous system. Megalin expression is reduced in diseases associated with fibrosis, including diabetic nephropathy, hepatic fibrosis and cholelithiasis, as well as in some breast and prostate cancers. One of the hallmarks of these conditions is the presence of the cytokine transforming growth factor beta (TGF-ß). Although TGF-ß has been implicated in the reduction of megalin levels, the molecular mechanism underlying this regulation is not well understood. Here, we show that treatment of two epithelial cell lines (from kidney and gallbladder) with TGF-ß1 is associated with decreased megalin mRNA and protein levels, and that these effects are reversed by inhibiting the TGF-ß1 type I receptor (TGF-ßRI). Based onin silicoanalyses, the two SMAD-binding elements (SBEs) in the megalin promoter are located at position...

Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite ...

The transcription factor Pax7 regulates skeletal muscle stem cell (satellite cells) specification and maintenance through various mechanisms, including repressing the activity of the muscle regulatory factor MyoD. Hence, Pax7-to-MyoD protein ratios can determine maintenance of the committed-undifferentiated state or activation of the differentiation program. Pax7 expression decreases sharply in differentiating myoblasts but is maintained in cells (re)acquiring quiescence, yet the mechanisms regulating Pax7 levels based on differentiation status are not well understood. Here we show that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4. Our results indicate that Nedd4 is expressed in quiescent and activated satellite cells, that Nedd4 and Pax7 physically interact during early muscle differentiation—correlating with Pax7 ubiquitination and decline—and that Nedd4 loss of function prevented this effect. Furthermore, even transient nuclear accumulation of Nedd4 induced a ...

Background Satellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin–proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined. Results Using conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers...