Microcystin-leucine arginine causes blood-testis barrier disruption and degradation of occludin mediated by matrix metalloproteinase-8

Yabing Chen1,2·Jing Wang1,2·Chun Pan1,2·Dongmei Li1,2·Xiaodong Han1,2

1Immunology and Reproduction Biology LaboratoryState Key Laboratory of Analytical Chemistry for Life Science, Medical School, Nanjing University, Nanjing 210093, China

2 Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing 210093, China

Y.B. Chen and J. Wang contributed equally to this manuscript,

Corresponding authors: Dongmei Li and Xiaodong Han

Email address: (D.L.) and (X.H.)

Tel.: +86 25 83686497

Supplementary Table S1.Specifications of primary antibodies

Vendor / Antibody / Catalog no. / Working dilution
Abcam / Rabbit anti-p-NF-B / Ab86299 / WB, 1:2000
Rabbit anti-GAPDH / Ab181602 / WB,1:5000
Rabbit anti-MMP-8 / Ab81286 / WB,1:1000 IF, 1:50
Boster / Rabbit anti-MMP-2 / BA0569 / WB,1:250
Rabbit anti-MMP-9 / PB0710 / WB,1:250
Cell Signaling / Rabbit anti-NF-B / #6956 / WB,1:1000 ChIP, 1:50
Rabbit anti-p-AKT / #4060 / WB,1:2000
Rabbit anti-AKT / #4691 / WB,1:1000
Rabbit anti-ERK / #4695 / WB,1:1000
Rabbit anti-p-ERK / #4370 / WB,1:2000
Rabbit anti-p-JNK / #4668 / WB,1:1000
Rabbit anti-JNK / #9252 / WB,1:1000
Rabbit anti-p-c-Jun / #3270 / WB,1:1000
Rabbit anti-c-Fos / #2250 / WB,1:1000 ChIP,1:50
Bioss / Rabbit anti-c-Jun / bs-0670R / WB,1:800
Thermo / Mouse anti-occludin / 33-1500 / WB,1:500 IF,1:200 IP,1:50

Fig. S1 Protein phosphatase 2A (PP2A) and reactive oxygen species (ROS)mediate downstream signaling pathways in response to MC-LR stimulation and induce matrix metalloproteinase-8 (MMP-8) expression in Sertoli cells (SC). a, bSC were pretreated with thePP2A activator D-erythro-sphingosine (DES) at 50 nM (a) or ROS scavenger N-acetylcysteine (NAC) at 5 mM (b) for 1 h, followed by a 24-h treatmentwith 500 nM MC-LR. The expression levels of phosphorylated AKT (p-AKT), total AKT, phosphorylated ERK (p-ERK), total ERK, phosphorylated JNK (p-JNK), and total JNK were detected by western blotting

Fig. S2MC-LR induced decreased expression of miRNA-184-3p in Sertoli cells (SC). SC were treated with MC-LR for 24 h, and the expression of miR-184-3p was determined by qRT-PCR. Data were expressed as means ± SEM (n=5). *p < 0.05 vs control.

Fig. S3miR-184-3p was significantly elevated in Sertoli cells (SC) and testis transfected with LV-miR-184-3p. a SC transfected with LV-miR-184-3p, LV-NC, LV-miR-184-3p-inhibitor, or LV-NC-inhibitor were treated with 500 nM MC-LR for 24 h; mRNA levels of miR-184-3p were examined by qRT-PCR. Data were expressed as means ± SEM (n=5). *p < 0.05. b Mice received LV-miR-184-3p, LV-NC, LV-miR-184-3p-inhibitor, or LV-NC-inhibitor via efferent duct injection, with intraperitoneal administration of 15 μg/kg body weight MC-LR. The mRNA levels of miR-184-3p were determined by qRT-PCR. Data were expressed as means ± SEM (n=5). *p < 0.05.

Fig. S4Up-regulation of miR-184-3p has no effects onmatrix metalloproteinase-8 (MMP-8)mRNA levels in testis. Mice received LV-miR-184-3p, LV-NC, LV-miR-184-3p-inhibitor, or LV-NC-inhibitor via efferent duct injection. One week later, mice were intraperitoneally injected with MC-LR at 15 μg/kg body weight for one week. The mRNA levels of miR-184-3p were determined by q-PCR. Data were expressed as means ± SEM (n=5). *p < 0.05.

Fig.S5Forced expression of miR-184-3p suppresses MC-LR-induced blood-testis barrier (BTB) destruction in mice. Mice received LV-miR-184-3p or LV-miR-184-3p-inhibitor via efferent duct injection.One week later, mice were intraperitoneally injected with MC-LR at 15 μg/kg body weight for one week.Electron microscope was used to examine the ultrastructural changes of BTB. Upper pictures indicate the typified structure of BTB (scale bar = 2 μM). Lower pictures correspond to magnified boxed areas (scale bar = 0.8 μM). The BTB is indicated by opposing white arrowheads. Basal ES, basal ectoplasmic specialization; ER, endoplasmic reticulum. White arrows indicate tight junction (TJ). Black arrowheads indicate actin filament bundle. The black asterisks indicate disassembly of the TJ.

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