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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Neurourol Urodyn. 2019 Aug 27;38(8):2140–2150. doi: 10.1002/nau.24152

The Effects of Microenergy Acoustic Pulses on animal model of Obesity-associated Stress Urinary Incontinence. Part 2: in situ Activation of Pelvic Floor and Urethral Striated Muscle Progenitor Cells

Ning Kang 1,3,#, Dongyi Peng 1,2,#, Bohan Wang 1, Yajun Ruan 1, Jun Zhou 1, Amanda B Reed-Maldonado 1, Lia Banie 1, Guifang Wang 1, Nianzeng Xing 3, Yuxin Tang 4, Guiting Lin 1, Tom F Lue 1,*
PMCID: PMC6801020  NIHMSID: NIHMS1046326  PMID: 31452249

Abstract

Aim:

To investigate the possibility and mechanism of microenergy acoustic pulses (MAP) for activating tissue resident stem/progenitor cells within pelvic and urethral muscle and possible mechanism.

Methods:

The female Zucker Lean and Zucker Fatty rats were randomly divided into four groups: ZL control, ZLMAP, ZF control and ZFMAP. MAP was applied at 0.033mJ/mm2, 3 Hz for 500 pulses, the urethra and pelvic floor muscles of each rat were then harvested for cell isolation and flow cytometry assay. Freshly isolated cells were analyzed by flow cytometry for Pax7, Int-7α, H3P and EdU expression. Meanwhile, pelvic floor muscle derived stem cells (MDSCs) were harvested through magnetic-activated cell sorting (MACS), MAP was then applied to MDSCs to assess the mechanism of stem cell activation.

Results:

Obesity reduced EdU-label retaining cells (EdU-LRC) and satellite cells in both pelvic floor muscle and urethra, while MAP activated those cells and enhanced cell proliferation, which promoted regeneration of striated muscle cells of the pelvic floor and urethra sphincter. Activation of Focal adhesion kinase (FAK)/ AMP-activated protein kinase (AMPK) /Wnt/β-catenin signaling pathways by MAP is the potential mechanism.

Conclusions:

MAP treatment activated tissue resident stem cells within pelvic floor and urethral muscle in situ via activating FAK-AMPK and Wnt/β-catenin signaling pathway.

Keywords: Obesity-associated stress urinary incontinence, microenergy acoustic pulses, endogenous stem cells, stem cell activation, cellular signaling pathway

1. INTRODUCTION

Our recent study indicates that obesity actually impairs the contractile properties of the urethral sphincter through intramyocellular lipid deposition, which leads to atrophy and distortion of the urethral striated muscle, an important contributor to obesity associated-SUI(OA-SUI)1,2. Current options for treatment of OA-SUI include oral medications, urethral bulking agents, and urethral sling surgeries, however, limited efficacy or short-term and long-term complications may occur3,4. Recently, exogenous stem cells have been applied to treat SUI-associated neuronal and muscular deficiencies5–8, but bear some negative side effects. Actually, the tissue resident adult stem cells (ASCs) within pelvic and urethral striated muscle tissue are potential target for SUI therapy. Modulating ASCs for tissue regeneration is eagerly needed, as well as the methods. In regulating stem cell/progenitor cell renewal and maintenance in a variety of systems and tissues, the Wnt/β-catenin signaling pathway play important role9. Researches demonstrated that activation of Wnt/β-catenin signaling enhances myogenesis10, increases myoblast proliferation, and enhances muscle growth11,12, whereas it has been reported that Wnt/β-catenin signaling can be activated by mechanotransduction13,14.

Low-intensity extracorporeal shockwave therapy (Li-ESWT) is one example of a mechanical force that has been employed for many years to treat musculoskeletal disorders15, ischemic heart disease16,17, and vasculogenic ED18,19. We previously reported that Li-ESWT activated penile stem cells20, 21 and very recently, we have applied Li-ESWT effectively to improve SUI22. In aims to clarify the mechanism, we have developed a similar but better technology -- microenergy acoustic pulses (MAP) -- which consists of a single predominantly positive pressure pulse followed by a relatively larger stretched wave component. Compared to standard shock wave, the MAP has a lower peak pressure (up to 21.8 mpa), slower pressure rises (5 milliseconds), and longer duration (~15 milliseconds). Most importantly, the beam of the acouostic pulses is defocused to give the treated tissue more even energy distribution and eliminate the potential damage to tissue at focal point if a focused device is used.

In current study, muscle derived stem/progenitor cells (MDSCs) from urethral and pelvic floor muscles post MAP were isolated and assayed with Flow cytometry, while the mechanism about MDSCs activation by MAP was also investigated.

2. MATERIALS & METHODS

2.1. Experimental design

Experiments were approved by the Institutional Animal Care and Use Committee at our University. Ten female Zucker Lean (ZL) rats (ZUC- Leprfa 186) and 10 Zucker Fatty (ZF) rats (ZUC-Leprfa 185) were used in this study. To track putative endogenous stem cells, newborn baby rats were received an intraperitoneal injection of 5-ethynyl-2-deoxyuridine (EdU; Invitrogen, Carlsbad, CA, USA. 150 mg/kg) at birth, as previously described23. Rats were divided into four groups (ZL control, ZLMAP, ZF control, and ZFMAP), each group including 5 rats. At 8 weeks old, MAP was applied as previously reported22. In brief, after application of ultrasound gel (Aquasonic, Parker Laboratories, Inc, Fairfield, NJ, USA), a special probe attached to a compact electromagnetic unit with a unfocused acoustic pulses source (LiteMed Inc, Taipei, Taiwan) was placed in contact with the pelvic region to include the urethra and the pelvic floor muscle in the treatment zone. Based on our previous experiments, we chose an acoustic energy of 0.033mJ/mm2, 3 Hz for 500 pulses, twice a week for 2 weeks24.

2.2. Cell isolation and Flow Cytometry Assay

Twenty-four hours post the last MPA treatment, the urethra and pelvic floor muscles (pubococcygeus and iliococcygeus muscles) of each rat were harvested for cells isolation following by flow cytometry. The isolated cells were isolated as previously reported25 and incubated with primary antibodies conjugated with Alexa 488-azide including Pax7(AB-528428, Tokyo Institute of Technology), Integrin α−7(sc-81807, Santa Cruz Biotechnology, Inc.), and H3P (EMD Millipore Corporation, CA, USA). They were then incubated with Click-iT reaction cocktail conjugated with Alexa594-azide (EdU Click-iT Cat# C10339; Invitrogen) for 30min at room temperature. Thereafter, the cells were analyzed in a fluorescence-activated cell sorter (FACS Vantage SE System; BD Biosciences) and FlowJo software (Tree Star, Inc., Ashland, OR.).

2.3. Treatment of muscle derived stem cells (MDSCs) with MAP in vitro

The pelvic floor muscle derived stem cells were isolated as previously reported25. To confirm that MAP is able to activate stem cells, the time and dosage response of MDSCs to MAP were assayed. For time response, MDSCs were treated with MAP at 0.033mJ/mm2,1Hz, 50 pulses then proteins were isolated at 1, 2, 4, 6 and 8 hours after treatment. For dosage response, MDSCs were treated with MAP at 0.033mJ/mm2,1Hz with 25, 50, 100, or 200 pulses followed protein isolation 4 hours post treatment (Fig S1). To evaluate whether MAP actives stem cells through the Wnt/β-catenin signaling pathway, FH535 (Wnt/β-catenin inhibitor) was used. Meanwhile, expression of EdU and H3P in MDSCs affected by MAP were checked with immunofluorescence (IF) staining as previously reported26. All experiments were repeated in triplicate and data were presented as the average of the three independent experiments.

2.4. Western blot

The western blot was conducted as previously reported27,26. Detection of target proteins on the membranes was performed with an electrochemiluminescence kit (Amersham Life Sciences Inc, Arlington Heights, IL) by using primary antibodies of p-FAK (1:500), FAK (1:500), p-AMPK (1:500), AMPK (1:300) (Cell Signaling Technology, Beverly, MA), β-catenin (1:500), cyclin D1 (1:500), and β-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA). After the incubation with secondary antibodies, the resulting images were analyzed with ChemiImager 4000 (Alpha Innotec) to determine the integrated density value of each protein band.

2.5. Statistical analysis

Data were analyzed with Prism 5 (GraphPad Software, San Diego, CA) and presented as means ± standard deviation (S.D). Statistical significance between two groups was analyzed by the Student t-test. For statistical significance among multiple groups, one-way ANOVA analysis followed by Bonferroni post hoc analysis was performed.

3. RESULTS

3.1. Obesity impaired EdU-label retaining cells (EdU-LRC) and satellite cells in pelvic floor muscle and urethra

EdU-LRC in both pelvic floor muscle and urethra were decreased in ZF rats as compared with ZL rats. The percentage of EdU-LRC was 0.86±0.06% in urethra and 0.75±0.07% in pelvic floor muscle in ZL and it decreased significantly to 0.48±0.03% (p< 0.01) in urethra and 0.46±0.05% (p< 0.01) in pelvic floor muscle in ZF group, respectively. The Pax-7 positive cells in ZL pelvic floor muscle was 0.39±0.11% and decreased to 0.19±0.02% (p< 0.01) in ZF, while the decrease in urethra is slight from 0.35±0.06% to 0.28±0.03% (P>0.05). Meanwhile, in ZF rats the Integrin α−7 positive cells were decreased significantly to 0.17±0.04% compared with ZL group 0.59±0.05% (p< 0.01) in pelvic floor muscle and also slightly decreased from 0.41±0.12% to 0.36±0.03% (P>0.05) in urethra (Fig 1).

Figure 1. Obesity impaired both EdU-LRC and satellite cells in pelvic floor muscle and urethra.

Figure 1.

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In pelvic floor muscle, obesity decreased EdU-LRC (**P< 0.01), integrin α−7 (**P< 0.01) and Pax-7 (**P< 0.01) significantly. In the urethra, obesity decreased the EdU-LRC (**P< 0.01) significantly and decreased the Integrin α−7 and Pax-7-positive cells slightly.

3.2. MAP activates EdU-LRC in pelvic floor muscle and urethra of both ZL and ZF rats

In the urethra, MAP significantly increased EdU-LRC from 0.86±0.06% to 1.86±0.16% (p< 0.01) in ZL and from 0.48±0.03% to 1.72±0.21% (p< 0.01) in ZF. Meanwhile, in the pelvic floor muscle, MAP also significantly increased EdU-LRC from 0.75±0.07% to 1.27±0.13% (p< 0.01) in ZL and from 0.46±0.05% to 1.37±0.13% in (p< 0.01) in ZF (Fig 2), which indicated ZF rats response better to MAP treatment.

Figure 2. MAP activates EdU-LRC in pelvic floor muscle and urethra.

Figure 2.

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MAP increased EdU-LRC within pelvic floor muscle and urethra both in ZL and ZF rats; MAP activated more EdU-LRC in ZF rats as compared with ZL rats in both pelvic floor muscle and urethra (**P< 0.01).

3.3. MAP activates satellite cells in pelvic floor muscle and urethra

To define muscle satellite cells, Integrin α−7 and Pax-7 were checked with FACS. MAP significantly increased Integrin α−7 positive cells in the urethra from 0.41±0.12% to 0.82±0.09% (p< 0.01) in ZL and from 0.36±0.03% to 0.95±0.12% (p< 0.01) in ZF. At the same time, MAP also significantly increased Pax-7 positive cells from 0.35±0.06% to 0.89±0.06% (p< 0.01) in ZL and from 0.28±0.03% to 1.12±0.21% (p< 0.01) in ZF (Fig 3).

Figure 3. MAP activates satellite cells in pelvic floor muscle and urethra.

Figure 3.

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MAP increased Integrin α−7 and Pax-7 positive cells in pelvic floor muscle and urethra in both ZL and ZF rats; MAP activated more Integrin α−7 positive cells in ZF rats as compared with ZL rats in pelvic floor muscle (**P< 0.01) but only slightly increased Integrin α−7 positive cells in urethra. MAP activated more Pax-7 positive cells in ZF rats as compared with ZL rats in both pelvic floor muscle (**P< 0.01) and urethra (*P< 0.05).

Interestingly, the situation was similar in the pelvic floor muscle, MAP significantly increased integrin α−7 positive cells from 0.59±0.05% to 1.35±0.27% (p< 0.01) in ZL and from 0.17±0.04% to 0.84±0.12% (p< 0.01) in ZF; and Pax-7 positive cells from 0.39±0.11% to 1.01±0.22% (p< 0.01) in ZL and from 0.19±0.02% to 1.01±0.18% (p< 0.01) in ZF (Fig 3). This indicated that MAP activated more satellite cells both in the urethra and in the pelvic floor muscle in ZF group.

3.4. MAP increased Cell proliferation in pelvic floor muscle and urethra

To check the cell proliferation in pelvic floor muscle and urethra, we assessed the number of cells staining positive for H3P, a nuclear protein indicative of cell proliferation. There are less H3P positive cells in both pelvic floor muscle and urethra in ZF compared with ZL. The H3P positive cells was 4.85±0.39% in urethra and 6.55±0.39% in pelvic floor muscle in ZL, while it decreased significantly to 3.93±0.35% (p< 0.01) in urethra and 4.43±0.42% (p< 0.01) in pelvic floor muscle in ZF (Fig 4a). MAP enhanced H3P positive cells in both the ZF MAP and ZL MAP groups as compared to their relative control (Fig 4b). MAP significantly increased H3P positive cells in the urethra to 15.02±0.75% in ZL and 14.98±0.71% in ZF (p< 0.01). In the pelvic floor muscle, MAP significantly increased H3P positive cells to 13.9±1.58% in ZL and 12.75±0.79% in ZF (p< 0.01). MAP activated more H3P positive cells in both urethra and pelvic floor muscle of ZF group.

Figure 4. MAP promotes cell proliferation in pelvic floor muscle and urethra.

Figure 4.

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(a). Obesity decreased H3P positive cells significantly in both pelvic floor muscle (**P< 0.01) and urethra (**P< 0.01); (b). MAP increased H3P-positive cells in pelvic floor muscle and urethra both in ZL and ZF rats (**P< 0.01); (c). MAP can activate more H3P-positive cells in ZF rats as compared with ZL rats in both pelvic floor muscle (**P< 0.01) and urethra (*P< 0.05).

3.5. MAP increased EdU-LRC differentiation into satellite cells

According to the results of flow cytometry, co-expression of EdU and Integrin α−7 (EdUIntegrin α−7 +) or Pax-7 (EdUPax−7+) among total EdU + cells were calculated, which provides the percentage of EDU-LRC that differentiate into satellite cells. In the pelvic floor muscle, the MAP increased EdUIntegrin α−7+ significantly from 0.582±0.059% to 0.683±0.091% in ZL (p < 0.05) and from 0.248±0.082% to 0.621±0.062% in ZF (p < 0.001). MAP increased EdUPax−7+ significantly from 0.419±0.129% to 0.587±0.095% in ZL (p < 0.05) and from 0.351±0.081% to 0.578±0.060% in ZF (p < 0.001) (Fig 5). In the urethra, MAP slightly increased EdUIntegrin α−7 + and EdUPax−7+ but not significantly (Figure S2).

Figure 5. MAP increases the percentage of EdU+ cells co-expressing Pax-7 and Integrin α−7 among the total EdU-LRC cells in pelvic floor muscle.

Figure 5.

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The percentage of EdU+ cells co-expressing Integrin α−7 and Pax-7 cell among the total EdU-LRC, abbreviated as EdUIntegrin α−7 +or EdUPax−7+. MAP significantly increased EdUIntegrin α−7 + and EdUPax−7+ in pelvic floor muscle of ZL rats (*P< 0.05) and ZF rats (**P< 0.01).

3.6. MAP activated MDSCs by modulating Wnt/β-catenin signal pathway

As we found that MAP promoted urethral and pelvic floor muscle cells proliferation characterized with activated H3P and more satellite cells in vivo, we assessed whether MAP could enhance H3P phosphorylation and EdU incorporation rate in MDSCs in vitro. Post the MAP treatment, the H3P increased to 15.29±0.51% from baseline of 4.81±0.51% (p < 0.001), while EdU incorporation rate increased to 59.73±2.93% from baseline of 49.29±2.45% (p < 0.05). Interestingly, this effect could be blocked by 5μMol/L FH 535 (Figure S3) which indicated Wnt signaling pathway might be involved in this biological effect.

Therefore, expression of cellular β-catenin after MAP was checked. We used different dosages MAP at 0.033 mJ/mm2,1Hz with 25, 50, 100, or 200 pulses to treat MDSCs. The results demonstrated that MAP increased the expression of β-catenin with a dosage dependent response and peaked at 50 pulses, which suggest 0.033 mJ/mm2, 1Hz,50 pulses of MAP is optimal for activation of Wnt signaling (Fig 6).

Figure 6. MAP activate the Wnt/β-catenin signal pathway in MDSCs.

Figure 6.

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(a). Six hours post MAP, more H3P positive cells (arrow head ***P< 0.001) and EdU positive cells (*P< 0.05) were noted with IF in the MAP group; (b). Four hours after treatment, expression of β-catenin increased start from 25 pulses of MPA (*P< 0.05) and peaked at 50 pulses (***P< 0.001).

3.7. MAP triggered the Wnt/β-catenin signal pathway through activation of FAK and AMPK.

It has been reported that AMP-activated protein kinase (AMPK) regulate the cellular Wnt signaling28,29, therefore, in current experiment, the AMPK pathway and its upstream Focal adhesion kinase (FAK) pathway were checked also. The phosphorylation level of FAK was 25.64±1.62% in control and increased by MAP treatment, peaked at 4 hours 46.32±2.03% (p < 0.01). Interestingly, AMPK phosphorylation of AMPK presented in a similar pattern. Expression of Cyclin D1 and β-catenin was also enhanced and peaked at 8 hours post MAP, which is later then the response of FAK and AMPK (Fig 7). The result indicated that MAP enhanced the phosphorylation of both FAK and AMPK, which led to the accumulation of β-catenin and activated the Wnt/β-catenin signal pathway, and subsequent expression of Cyclin D1.

Figure 7. MAP activates the Wnt/β-catenin signal pathway through FAK and AMPK.

Figure 7.

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(a). Post MAP treatment, the phosphorylation of FAK was increased from 1hr (**P< 0.01) and peaked at 4hrs (**P< 0.01), then slightly dropped at 6hrs (**P< 0.01). The phosphorylation of AMPK was also increased in similar pattern. Expression of β-catenin started to increase from 1hr post MAP and peaked at later phase of 8hrs (*P< 0.05) while Cyclin D1 has the similar later-phase pattern (*P< 0.05; **P< 0.01; ***P< 0.001); (b). Hypothetical mechanism for MDSC activation by MAP.

4. DISCUSSION

Many studies have shown that obesity is associated with more frequent episodes of urinary incontinence and a greater prevalence of SUI30. Subak et al. documented a clear dose-response effect of weight on urinary incontinence with each 5-unit increase in BMI associated with a 20–70% increase in incontinence risk31,32. However, exact mechanisms of obesity associated stress urinary incontinence (OA-SUI) are not clear. Our previous study demonstrated that obesity can lead to intra-myocellular lipid deposition in the urethral sphincter and to atrophy and distortion of urethral striated muscle1,2.

Recently, cell-based therapies have been developed and may have therapeutic applications for OA-SUI. In 2010, we applied adipose derived stem cells (ADSC) for the treatment of SUI in an animal model and many therapeutic challenges were identified at that time8. However, the disadvantage of exogenous SCs were identified and obviously33. In situ activation of tissue-resident satellite cells within the urethral and pelvic floor muscle is an exciting potential therapy for OA-SUI that avoids these potentially significant negative consequences of exogenous stem cell therapy. Evident demonstrated that mechanical forces direct tissue development, morphogenesis, and regeneration34, while low intensity extracorporeal shock wave therapy (Li-ESWT), as a mechanical force, activated penile stem cells20 and increased phosphorylated histone 3 (H3P) level21. Very recently, we reported that MAP effectively improved vaginal distension induced SUI22. We also sucessfully applied MAP to an OA-SUI rat model and improved the stress incontinence (In manuscript Part I).

To explore the mechanism of MAP in treating OA-SUI, in current study, MAP was applied to pelvic floor and urethra and check it biological effect on EdU-LRC and satellite cells within. Results indicated the EdU-LRC and muscle satellite cells were impaired in both pelvic floor muscle and urethra tissue in obesity ZF rats. Strikingly, MAP significantly reversed this condition, as shown in Figure 2, the total number of EdU-LRC, integrin α−7 and Pax-7 + cells were markedly enhanced by MAP treatment. Moreover, we found ZF rats have a more robust response to MAP than ZL. Importantly, as shown in Figure 5, the EdUIntegrin α−7 + and EdUPax−7+ were increased in both ZL and ZF rats which means MAP promoted EdU-LRC differentiation into muscle satellite cells and further into mature muscle cells for tissue regeneration.

Multiple studies employing different experimental models suggest that a complex and dynamic cross-talk exists between the FAK and Wnt/β-catenin signaling pathways35,36. Mechanical stimulation leads to FAK activation which phosphorylates GSK3β and stabilizes β-catenin protein to promote its nuclear translocation and activation of target-gene expression35,36. There are some studies37–39 showing that shock-wave therapy can promote the phosphorylation of FAK. In our current study, MAP significantly increased both β-Catenin and cyclin D1 which could be blocked by FH535, a Wnt signaling inhibitor. Means while, the ratio of p-FAK/FAK in MDSCs was increased and peaked at 4 hours after MAP treatment.

Recently, it was shown that AMPK can cross-talk with Wnt/β-catenin, promoting β-catenin expression through phosphorylation of HDAC5 and phosphorylating β-catenin at Ser 552, which stabilizes β-catenin and enhances β-catenin/TCF- mediated transcription28,29. In curent study, it was found that MAP activate both FAK and AMPK signaling pathways followed activation of Wnt signaling pathways and increase of Cyclin D1 and H3P. However, whether FAK and AMPK play a dominant role in MAP-Wnt signal pathway activation remains to be further studied.

5. CONCLUSIONS

Cuurent results indicate that obesity produces a detrimental effect on the tissue resident stem cells and impaired tissue regeneration. MAP activated both EdU-LRC and satellite cells and promoted the regeneration of striated muscle cells within pelvic floor and urethra sphincter. Activation of FAK /AMPK/Wnt/β-catenin signaling pathways by MAP is the potential mechanism. Despite those promising findings, it should be cautioned that current study has limitations and further validation is needed, while the exact mechanisms of mechano-activation of endogenous stem/progenitor cells by MAP should be explored.

Supplementary Material

Supp figS1-3

Figure S1. Schematic graph of MAP treatment on MDSCs. 2 days after cells seeding in the plate, different dosage of MAP(0.033mJ/mm2,1Hz with 25, 50, 100, or 200 pulses) were used to treat the cells.

Figure s2. MAP increases the percentage of EdU-LRC co-express Pax-7/Integrin α−7 cells in urethra. The percentage of EdU co-express Integrin α−7/Pax-7 cell abbreviate as EdUIntegrin α−7 or EdUPax−7+. MAP slightly increased EdUIntegrin α−7 + and EdUPax−7+in the urethra of ZL and ZF rats (P>0.05).

Figure s3. MAP promoted MDSCs proliferation vs Wnt signaling pathway. Six hours post MAP treatment, EdU (Geen) incorporation and H3P (Red, arrow head) expression were significantly enhanced in MDSCs and blocked by 5uM FH535. (*P< 0.05, ***P< 0.001)

ACKNOWLEDGMENTS:

Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number R56DK105097 and 1R01DK105097. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Associated Data

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Supplementary Materials

Supp figS1-3

Figure S1. Schematic graph of MAP treatment on MDSCs. 2 days after cells seeding in the plate, different dosage of MAP(0.033mJ/mm2,1Hz with 25, 50, 100, or 200 pulses) were used to treat the cells.

Figure s2. MAP increases the percentage of EdU-LRC co-express Pax-7/Integrin α−7 cells in urethra. The percentage of EdU co-express Integrin α−7/Pax-7 cell abbreviate as EdUIntegrin α−7 or EdUPax−7+. MAP slightly increased EdUIntegrin α−7 + and EdUPax−7+in the urethra of ZL and ZF rats (P>0.05).

Figure s3. MAP promoted MDSCs proliferation vs Wnt signaling pathway. Six hours post MAP treatment, EdU (Geen) incorporation and H3P (Red, arrow head) expression were significantly enhanced in MDSCs and blocked by 5uM FH535. (*P< 0.05, ***P< 0.001)

NIHMS1046326-supplement-Supp_figS1-3.pdf (824.3KB, pdf)

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