Ultrasonography of Osteosarcoma in Dogs
Sonography Atlas of Dogs

Abstract

To quantitatively measure the effects of treatment on STAT3 expression, canine OSA cells (OSA8) were treated with curcumin or FLLL32 for 4 or 24 hours, and RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. cDNA was made from 1 μg total RNA using the Superscript III kit (Invitrogen). Real-time quantitative PCR was performed using the Applied Biosystem's StepOne Plus Real-Time PCR System. STAT3 and 18S mRNA were detected using Fast SYBR green PCR master mix (Applied Biosystems) according to the manufacturer's protocol and primer sets are detailed in Table 2. All reactions were performed in triplicate and included no-template controls for each gene. Relative expression was calculated using the comparative threshold cycle method [45]. Experiments were repeated 3 times using samples in triplicate.

Table 2: Primers for STAT3 quantitative reverse transcriptase polymerase chain reaction

Results

Treatment with curcumin or FLLL32 decreased proliferation of OSA cell lines

Canine (OSA8, 16, and D17) and human (SJSA and U2OS) OSA cell lines were treated with 10 μM curcumin or increasing concentrations of FLLL32 for 72 hours and proliferation was measured. Figure Figure1A1A shows that both canine and human OSA cell lines exhibited significant decreases in proliferation after treatment with FLLL32, particularly at concentrations above 0.75 μM. Interestingly, while the human cell lines were sensitive to curcumin treatment, the canine lines appeared to be somewhat resistant. However, FLLL32 induced a statistically significant greater effect on proliferation of all OSA cell lines at lower concentrations (2.5 μM and 7.5 μM) when compared to that induced by curcumin at 10 μM. As depicted in Figure Figure1B,1B, the IC₅₀ for FLLL32 ranged from 0.75-1.45 μM for the OSA cell lines as extrapolated from logarithmic curves. These data demonstrate that FLLL32 is more potent than curcumin, with FLLL32 inhibiting cell proliferation at lower concentrations than curcumin both in canine and human OSA cell lines.

Figure 1: Treatment with curcumin or FLLL32 decreased the proliferation of OSA cell lines. A) Canine (OSA8, OSA16, and D17) or human OSA cell lines (SJSA and U2OS) were treated with vehicle, curcumin, or FLLL32 for 72 hours. Proliferation was analyzed using the CyQUANT^® cell proliferation assay kit. Proliferation values are listed as a percentage of DMSO control. Experiments were performed in quadruplicate and repeated two times. Statistical analysis of cell proliferation was performed using the Student's t-test. P values of < 0.05 were considered statistically significant.*p < 0.05 B) Canine and human OSA cell lines were treated with DMSO or FLLL32 for 72 hours and analyzed with CyQUANT^® to determine proliferation as a percentage of DMSO control. IC₅₀s for FLLL32 were calculated for each cell line using a log curve. Experiments were performed in quadruplicate and repeated two times.

FLLL32 induced activation of caspase 3/7, PARP cleavage, and apoptosis of OSA cell lines

Previous work in our laboratory demonstrated that siRNA mediated downregulation of STAT3 expression in human and canine OSA cell lines induced apoptosis [14]. To evaluate the effects of FLLL32 on OSA cells, canine and human OSA cell lines were cultured with curcumin or increasing concentrations of FLLL32 for 24 hours and apoptosis was measured. Significant increases in caspase 3/7 activity occurred at 7.5 μM of FLLL32 compared to curcumin at 10 μM (Figure 2A). Additionally, we examined the status of poly (ADP-ribose) polymerase (PARP), a nuclear enzyme important for chromosomal structure and genomic stability [46]. PARP cleavage occurs following caspase-3 activation during the process of apoptosis [47]. A dose-dependent increase in PARP cleavage (cPARP) in both canine and human OSA cell lines also occurred after 24 hours of treatment with FLLL32 (Figure 2B). In contrast, there was minimal to no PARP cleavage induced by treatment with 10 μM curcumin (Figure 2B).

Figure 2: FLLL32 induced activation of caspase 3/7, PARP cleavage, and apoptosis of OSA cell lines. A) Canine (OSA8, OSA16, and D17) and human (SJSA and U2OS) OSA cell lines were treated with media, DMSO, curcumin, or FLLL32 for 24 hours. Apoptosis was assessed by measuring active caspase-3/7 using the SensoLyte^® Homogeneous AMC Caspase-3/7 Assay kit. Experiments were performed in triplicate and repeated two times. Statistical analysis of the caspase 3/7 activity was performed using the Student's t-test. P values of < 0.05 were considered statistically significant.*p < 0.05 B) Canine (OSA8, OSA16, and D17) or human (SJSA and U2OS) OSA cell lines were treated with DMSO, curcumin, or FLLL32 for 24 hours prior to collection. Protein lysates were generated and separated by SDS-PAGE and western blotting for PARP and β-actin was performed. Experiments were repeated two times.

FLLL32 decreased STAT3 DNA binding in OSA cell lines

The curcumin analog FLLL32 acts in part through direct inhibition of STAT3 DNA binding by interacting with its SH2 domain, which is critical for dimerization [38]. We observed that both canine (OSA8) and human (SJSA) OSA cells exhibited decreased STAT3 DNA binding after only 4 hours of treatment with curcumin or FLLL32 (Figure 3A). To determine if the decrease in DNA binding was due to loss of STAT3 total protein, we harvested protein from cells concurrently treated for 4 hours and observed no significant decrease in STAT3 protein compared to media or DMSO- treated cells (Figure 3B).

Figure 3: FLLL32 decreased STAT3 DNA binding in OSA cell lines. A) Canine OSA cell line OSA8 and human OSA cell line SJSA were incubated with media, DMSO, 10 μM curcumin, or 10 μM FLLL32 for 4 hours. Cells were harvested and nuclear protein isolated. Nuclear protein was added to binding reactions with labeled species specific DNA probes for the STAT3 recognition sequences located in the promoter for survivin in the presence or absence of unlabelled competitor probe. Additionally, anti-STAT3 antibody was added to nuclear protein from cells treated with media alone to demonstrate specificity of the binding reaction. Reactions were separated on an acrylamide gel, transferred to a nylon membrane, and the DNA was crosslinked. The membranes were processed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific Inc, Rockford, IL). Experiments were repeated two times. B) Canine OSA cell line OSA8 and human OSA cell line SJSA were incubated with media, DMSO, 10 μM curcumin, or 10 μM FLLL32 for 4 hours concurrently with cells treated for EMSA above. Cells were harvested and total protein isolated and quantified. Protein was separated by SDS-PAGE. Western blotting was performed for STAT3 and β-actin. Experiments were repeated two times.

Downregulation of STAT3 via FLLL32 treatment decreased expression of VEGF, MMP2, and survivin

Given the role of survivin, VEGF, and MMP2 in tumor cell survival, angiogenesis, and metastasis, we determined if downregulation of STAT3 DNA binding correlated with loss of expression of these STAT3 transcriptional targets in OSA cell lines. Canine (OSA8) and human (SJSA) OSA cells were treated for 12 or 24 hours with DMSO, 10 μM curcumin, or 10 μM FLLL32. Loss of MMP2 mRNA expression occurred in OSA8 at both 12 and 24 hours after treatment with 10 μM FLLL32; however, loss of MMP2 mRNA in the SJSA line was not noted until 24 hours of FLLL32 exposure (Figure 4A). Treatment with 10 μM FLLL32 resulted in loss of VEGF mRNA expression in both cell lines after 24 hours of drug treatment (Figure 4A). Additionally, downregulation of VEGF protein expression was similarly observed following 24 hours of FLLL32 exposure at 10 μM and was also noted at lower concentrations (2.5 and 5 μM) of drug (Figure 4B). Interestingly, VEGF mRNA levels appeared to be increased in the OSA8 and SJSA lines after 24 hours of exposure to 10 μM curcumin, although this did not correlate with the observed changes in VEGF protein in which VEGF was unchanged (SJSA) or downregulated (OSA8) after curcumin treatment. Decreases in survivin expression occurred at 5 and 10 μM FLLL32 in the canine OSA lines and at 2.5 μM FLLL32 and higher in the human OSA lines (Figure 4B). Curcumin downregulated survivin expression in the human but not canine OSA lines, supporting the notion that, as with the previously discussed proliferation data, the human cells are much more sensitive to the effects of curcumin.

Figure 4: Downregulation of STAT3 via FLLL32 treatment decreased expression of VEGF, MMP2, and survivin. A) Canine (OSA8) or human (SJSA) OSA cell lines were treated with DMSO, 10 μM curcumin, or 10 μM FLLL32 for 12 or 24 hours. RNA was collected and RT-PCR was performed for VEGF, MMP2, and GAPDH. Experiments were repeated two times. B) Canine (OSA8, OSA16, and D17) or human (SJSA and U2OS) OSA cell lines were treated with DMSO, curcumin, or FLLL32 for 24 hours prior to collection. Protein lysates were generated and separated by SDS-PAGE and western blotting for survivin, VEGF, and β-actin was performed. Experiments were repeated two times.

Treatment with FLLL32 decreased pSTAT3 and total STAT3 expression in canine and human OSA

Human and canine OSA cells were treated with 10 μM curcumin or increasing concentrations of FLLL32 for 24 hours to determine their effect on STAT3 phosphorylation. There was a dose dependent decrease in STAT3 tyrosine 705 phosphorylation as demonstrated by Western blotting with downregulation occurring at 2.5 μM FLLL32 (Figure 5A). Additionally, decreases in total STAT3 occurred after FLLL32 treatment in all cell lines treated (Figure 5A). To determine the mechanism for loss of total STAT3 protein, we treated canine and human OSA cell lines with FLLL32 for 24 hours and performed RT-PCR to determine whether this was due to loss of stat3 gene expression as STAT3 is known to regulate its own expression through an autoregulatory loop [48]. Using standard RT-PCR, there was no downregulation of STAT3 mRNA expression after 24 hours with treatment with curcumin or FLLL32 (Figure 5B). When OSA8 cells were treated with FLLL32 and STAT3 expression was evaluated using quantitative real time PCR, a small decrease in STAT3 mRNA expression was present at 24 hours, but this was not statistically significant (Additional File 1) and therefore would be unlikely to account for the protein loss observed by western blotting. Lastly, the loss of STAT3 was not due to global loss of proteins secondary to cell death as there were no differences in the levels of pERK1/2 and total ERK 1/2 in OSA cell lines treated with drug for 24 hrs (Additional File 2).

Figure 5: Treatment with FLLL32 decreased pSTAT3 and total STAT3 expression in canine and human OSA cell lines. A) Canine (OSA8, OSA16, and D17) or human (SJSA and U2OS) OSA cell lines were treated with DMSO, curcumin, or FLLL32 for 24 hours prior to collection. Protein lysates were generated and separated by SDS-PAGE and western blotting for pSTAT3 (Y705), total STAT3, and β-actin was performed. Experiments were repeated two times. B) Canine (OSA8 and 16) or human (SJSA) OSA cell lines were treated with DMSO, 10 μM curcumin, or 10 μM FLLL32 for 24 hours. RNA was collected and RT-PCR was performed for STAT3 and GAPDH. Experiments were repeated two times. C) Canine OSA cell line OSA8 was treated with DMSO, curcumin, or FLLL32 for 24 hours prior to collection. Protein lysates were generated and separated by SDS-PAGE and Western blotting for ubiquitin, STAT3, and β-actin was performed. Experiments were repeated two times. D) OSA8 was treated with DMSO or FLLL32 for 4 hours prior to collection. Protein lysates were generated and STAT3 was immunoprecipitated. Protein was separated by SDS-PAGE and western blotting for ubiquitin and STAT3 was performed. Experiments were repeated two times.

STAT3 downregulation after FLLL32 treatment occurred through the ubiquitin/proteasome pathway

STAT family proteins are known to be regulated by ubiquitin-mediated degradation [49,50]. To determine if this mechanism was responsible for the loss of total STAT3 following FLLL32 treatment, the OSA8 cell line was treated with curcumin or FLLL32 for 24 hours and Western blotting for ubiquitin was performed on lysates. An intense band emerged at 75 kDa in FLLL32 treated cells corresponding to the size of STAT3 (Figure 5C). We next immunoprecipitated STAT3 and performed Western blotting for ubiquitin. A band was present at 75 kDa in addition to a smear directly above the band in the group treated with 10 μM FLLL32 for 4 hours (Figure 5D). This was interpreted to be mono-ubiquitinylated STAT3 at 75 kDa and poly-ubiquitinylated STAT3 protein at the large molecular weight sizes. Indeed, after treating OSA8 cells with curcumin, FLLL32, or the proteasome inhibitor MG132, there was almost a four-fold increase in poly-ubiquitinylated STAT3 in cells treated with FLLL32 as compared to those treated with curcumin (Figure 6A). Immunoblotting for β-actin was performed to confirm the specificity of the immunoprecipitation experiment; none was detected (data not shown). Although it has been reported that curcumin has proteasome inhibition properties [51], treatment with curcumin or FLLL32 did not lead to alteration in the activity of the 20S proteasome when compared with MG132 at the same concentration (Figure 6B).

Figure 6: Loss of STAT3 occurred in part through the ubiquitin-proteasome pathway however curcumin and FLLL32 did not inhibit the 20S proteasome in OSA cell lines. A) OSA8 was treated with DMSO, curcumin, FLLL32, or the proteasome inhibitor MG132 for 4 hours prior to collection. Protein lysates were generated and STAT3 was immunoprecipitated. Protein was separated by SDS-PAGE and western blotting for ubiquitin and STAT3 was performed. Experiments were repeated two times. Densitometry analysis was performed using Image J (Rasband, W. S., Image J, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2009). B) Canine (OSA8) or human (SJSA) OSA cells were serum starved for 2 hours then treated with DMSO, 10 μM curcumin, 10 μM FLLL32, or 10 μM MG132 for 4 hours. Cells were collected, washed with cold PBS, and prepared for use in the 20S Proteasome Activity Assay Kit (Millipore, Billerica, MA). Experiments were repeated two times.

Inhibition of caspase activation did not affect loss of STAT3 following FLLL32 treatment

Activated caspases 2, 4, 5, and 10 are known to be capable of cleaving STAT3 [52]. To investigate whether loss of STAT3 after treatment with FLLL32 was due to cleavage by activated caspases, we pretreated the OSA8 and SJSA cell lines with a pan-caspase inhibitor Z-VAD-FMK for 2 or 24 hours and then added FLLL32 or DMSO to the cells for an additional 18 hours. Western blotting of cell lysates demonstrated that inhibition of caspase activity by Z-VAD-FMK abrogated PARP cleavage but it did not significantly alter the amount of total STAT3 remaining after FLLL32 treatment compared with cells treated with FLLL32 and no Z-VAD-FMK (Figure 7A). Furthermore, Z-VAD-FMK pretreatment abrogated caspase 3/7 activation but this had no effect on the loss of STAT3 following FLLL32 treatment (Figure 7B). These data indicate that loss of STAT3 protein after FLLL32 exposure was not due to caspase- mediated cleavage.

Figure 7: Inhibition of caspase activation did not affect loss of STAT3 following FLLL32 treatment. A) Canine (OSA8) and human (SJSA) OSA cell lines were pretreated with the pan-caspase inhibitor 80 μM Z-VAD-FMK or DMSO for 2 or 24 hours then treated with DMSO or 10 μM FLLL32 for 18 hours. Protein lysates were generated and separated by SDS-PAGE and western blotting for STAT3, PARP, and β-actin was performed. Experiments were repeated two times. B) Canine (OSA8) or human (SJSA) OSA cell lines were pretreated with the pan-caspase inhibitor 80 μM Z-VAD-FMK, DMSO, or media for 2 or 24 hours then treated with media, DMSO, or 10 μM FLLL32 for 18 hours. Caspase-3/7 activity was measured using the SensoLyte^® Homogeneous AMC Caspase-3/7 Assay kit. Experiments were performed in triplicate and repeated two times.

Discussion

Curcumin has a long history of use as a medicinal compound and is known to have multiple anti-inflammatory and anti-cancer properties; however, blood levels that can be achieved after oral administration are low, which limits its potential clinical value [20,27,34]. Curcumin also affects a broad range of cellular targets including STAT3 [53,54] in addition to a host of other signaling molecules such as Wnt/β-catenin [26], NF-κB [20], and HER2 [27], and the proteasome [51]. Given the number of targets affected by curcumin and its poor bioavailability, efforts have been directed at improving its chemical properties by complexing it with lipids/phospholipids [55,56] and developing more specific derivatives [35,57-59]. Interestingly, many of these analogues have demonstrated greater stability and more potent activity against several tumor cell lines, including those derived from breast, prostate, pancreas, and colon cancers when compared to curcumin [35,57-59]. Curcumin has been found to be well-tolerated in healthy individuals and OSA patients [18], most recently when given as a solid lipid particle formulation. However, peak plasma levels reached only 22.43 ng/mL (approximately 60 nM), well below concentrations known to have biologic effects against OSA cells in vitro.

During the development of novel curcumin analogs, our collaborators determined that one of these compounds, FLLL32, was particularly effective at suppressing the growth of pancreatic and breast cancer cells [38]. To produce FLLL32, the two hydrogen atoms on the central carbon of curcumin were replaced with a spiro-cyclohexyl ring. It was proposed that this alteration would confer greater stability and specificity for STAT3 than curcumin [38]. Recent work with FLLL32 showed that it induced apoptosis in human melanoma, multiple myeloma, glioblastoma, pancreatic, breast, and colorectal cancer cell lines and inhibited STAT3 phosphorylation and DNA binding [38-40]. The compound also exhibited higher potency at inhibiting proliferation and STAT3 DNA binding activity than curcumin and other JAK/STAT3 inhibitors in human rhabdomyosarcoma cells [42]. Indeed, FLLL32 has been shown to be more potent than other STAT3 inhibitors in promoting growth inhibition of multiple cancer cell lines, and the drug is improved in its specificity as demonstrated by kinase profile assays that revealed almost no activity against tyrosine kinases such as Lck, Syk, Lyn, Yes, and Abl-1 [39]. Given the superior specificity and efficacy of FLLL32 as compared to curcumin in a variety of cancer cell lines, the purpose of this study was to evaluate the biologic activity of this compound against OSA cell lines.

Previous studies have explored the activity of curcumin against OSA both in vitro and in human clinical trials [18,20,27]. OSA cell lines experienced cell cycle arrest, reduced proliferation, and underwent apoptosis following treatment with curcumin [19,33,60]. Prior work in our laboratory demonstrated that STAT3 is constitutively activated in OSA cell lines and that inhibition of STAT3 through STAT3 siRNAs or the small molecule STAT3 inhibitor LLL3 resulted in loss of proliferation and apoptosis [14]. Data presented in this study showed that FLLL32 inhibited proliferation of OSA cell lines and promoted apoptosis via caspase 3/7 activation at lower concentrations than curcumin. This is consistent with recent work demonstrating apoptosis via caspase activation in human multiple myeloma, glioblastoma, liver cancer, colorectal, and melanoma cell lines after FLLL32 exposure [39,40]. Cleavage of PARP, an indicator of caspase-3-mediated apoptosis, was also seen in many of these human cancer cell lines upon treatment with FLLL32 [39]. Interestingly, loss of messenger RNA and protein expression of survivin, an inhibitor of apoptosis, as well as decreased STAT3 DNA binding activity was observed in human rhabdomyosarcoma cells treated with FLLL32 [42]. The concurrent reduction in STAT3 transcriptional activity of targets such as survivin through decreased DNA binding and loss of STAT3 phosphorylation likely both played a role in the reduced survival of OSA tumor cells observed following exposure to FLLL32.

Recent work has shown that expression of high levels of STAT3 in human OSA tumor samples correlated to poor differentiation, metastasis, and lower rates of overall and relapse-free survival [11]. Overexpression of phosphorylated STAT3 in OSA has also been linked to poor prognosis [17]. STAT3 is known to enhance tumor cell invasion, metastasis, and angiogenesis through enhanced expression of VEGF and MMP2 [61]. Human patients with OSA whose tumors had higher VEGF expression as shown by immunohistochemistry had a significantly worse prognosis and had lung metastasis [62,63]. Previous work revealed that treatment of OSA cell lines with curcumin inhibited their migration [26]. Mouse xenograft models of pancreatic and colorectal cancer treated with curcumin exhibited suppression of tumor angiogenesis and tumor growth inhibition [20]. In more recent studies, FLLL32 inhibited vascularity and tumor growth in chicken embryo xenografts and reduced tumor volume in mouse xenografts of breast cancer [38]. Our data demonstrate that in the OSA cell lines we tested, VEGF mRNA and protein and MMP2 mRNA were expressed and treatment with 10 μM FLLL32 downregulated the expression of these STAT3 transcriptional targets following 24 hours of drug exposure. Interestingly, VEGF mRNA expression appeared to increase over baseline in both the OSA8 and SJSA lines after curcumin exposure, although this did not correlate with the findings obtained by Western blotting in which VEGF protein was absent in OSA8 cells and unchanged in SJSA cells. The mechanism for this observed discrepancy is not clear, although there are several possible explanations. Curcumin may somehow interfere with translation of VEGF mRNA, directly enhance degradation of VEGF protein, or alternatively, given its diversity of cellular targets, affect proteins other than STAT3 that in turn alters VEGF expression. Further investigation of these potential mechanisms is needed. Given the putative role of both VEGF and MMP2 in the process of tumor growth and metastasis and recent data demonstrating the ability of FLLL32 to abrogate breast cancer xenograft growth in mice, future work assessing the effects of FLLL32 in mouse models of OSA is warranted.

Treatment of OSA cell lines with FLLL32 promoted loss of both pSTAT3 and total STAT3. This loss of STAT3 correlated with the presence of mono- and polyubiquitinylated STAT3, indicating that proteasome-mediated degradation was likely responsible for the observed decrease in protein. Interestingly, curcumin has been shown to inhibit activities of the proteasome in certain cancer cells [51]; however we detected no evidence for this activity after treating the OSA cell lines with curcumin or FLLL32 at the doses and time points examined. Although modulation of STAT3 protein levels is known to occur in part through caspase cleavage [52] a pan-caspase inhibitor did not affect the observed loss of STAT3 after FLLL32 treatment. Additionally, we did not see a significant decrease in STAT3 mRNA 24 hours after FLLL32 treatment, indicating that loss of STAT3 mRNA could not be primarily responsible for the protein downregulation that occurs after FLLL32 exposure. These data support the assertion that in addition to blocking STAT3 function, FLLL32 acts to promote downregulation of STAT3 protein, thereby enhancing the functional consequences of this small molecule inhibitor.

Conclusions

The novel small molecule STAT3 inhibitor FLLL32 downregulated proliferation and promoted apoptosis of OSA cells. FLLL32 inhibited STAT3 DNA binding and induced proteasome mediated degradation of STAT3 resulting in a subsequent loss of VEGF, MMP2, and survivin expression. These data support the notion that STAT3 is a relevant target for therapeutic intervention in OSA and that FLLL32 and similar analogs may have clinical utility for the treatment of OSA.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SF designed and carried out molecular experiments on OSA cell lines and drafted the manuscript. MB participated in RT-PCR design and performance. JL, CL, ES, PL, and JRF designed and produced the small molecule STAT3 inhibitor FLLL32. JF designed and carried out the quantitative real time PCR experiments. WK assisted in experimental design. CL conceived of the study, assisted in experimental design, and helped draft the manuscript. All authors read and approved the final manuscript.

Pre-publication history

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1471-2407/11/112/prepub

Supplementary Material

Curcumin and FLLL32 downregulated the expression of STAT3 protein without significant inhibition of STAT3 mRNA transcript levels. OSA8 cells were treated with 10 μM curcumin or 10 μM FLLL32 and were collected at 4 and 24 hours after treatment, and real-time PCR for STAT3 mRNA was performed. Bars represent STAT3 relative expression (2^-Delta Ct). Experiments were performed in triplicate and repeated three times. The difference between treatment groups and DMSO control group was analyzed using the Students t test. P values of < 0.05 were considered statistically significant. There was no statistical significance between the treatment groups.

Additional file 2:

Treatment with curcumin or FLLL32 did not significantly alter pERK1/2 or total ERK1/2 levels. Canine (OSA8) or human (SJSA) OSA cell lines were treated with DMSO, 10 μM curcumin, or increasing concentrations of FLLL32 for 24 hours prior to collection. Protein lysates were generated and separated by SDS-PAGE and western blotting for pERK1/2 (Thr202/Tyr204), total ERK1/2, and β-actin was performed. Experiments were repeated two times.

Acknowledgements and Funding

This work was supported by grants from the Morris Animal Foundation, the American Kennel Club, and NIH K01 (1K01RR025393-01). The authors would like to thank Tim Vojt of The Ohio State University College of Veterinary Medicine Biomedical Media Services for his assistance in figure preparation.

References

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