P4-induced aberrant lysosomal function is rescued by TRPML1.

(A and B) PK-15 cells were infected with PRV HN1201 (MOI = 0.1) for 0–24 hours. The mRNA levels of TPCN1 (A) and TPCN2 (B) were analyzed by qRT-PCR. (C) PK-15 cells were treated as indicated in (A). TPCN1 and TPCN2 proteins were analyzed by immunoblotting. (D) Mice were either mock-infected or intranasally infected with PRV (2 × 103 TCID50 per mouse) for 2 days. TPCN1 and TPCN2 in the uterus were analyzed by immunoblotting (n = 3). (E) PK-15 cells were transfected with TRPML1-FLAG, TRPML1-FLAG L106P, TPCN1-FLAG, and TPCN2-FLAG plasmids and treated with vehicle or P4 (10 μM) for 24 hours. Colocalization of LAMP1 with TRPML1 variants, TPCN1, and TPCN2 was analyzed by immunofluorescence analysis. Scale bar: 10 μm. (F) Quantification of the perinuclear index of LAMP1 in PK-15 cells from (E). (G) PK-15 cells were transfected with TRPML1-mCherry, TRPML1-mCherry L106P, TPCN1-mCherry, and TPCN2-mCherry and treated with vehicle or P4 (10 μM) for 24 hours. Colocalization of fluo-3 and intracellular cholesterol (filipin) with TRPML1 variants, TPCN1, and TPCN2 was analyzed by immunofluorescence. Scale bar: 10 μm. (H) TRPML1 in scramble, shTRPML1-1, and shTRPML1-2 was detected by immunoblotting. (I) PK-15 cells were treated with P4 (10 μM), ML-SA1 (20 μM), and ML-SI1 (10 μM) as indicated. Fluo-3 and filipin staining were performed 8 hours post-treatment. Scale bar: 10 μm. (J) PK-15 cells were incubated with PRV HN1201 (MOI = 0.1) and treated with vehicle, P4 (10 μM), ML-SA1 (20 μM), or ML-SI1 (10 μM) as indicated at 4°C for 2 hours. Cells were then shifted to 37°C for 10 minutes to allow entry. After washing with trypsin (1 mg/mL) to remove residual virions on the PM, viral entry was detected by qRT-PCR analysis of viral genome copy numbers in the cells. (K) PK-15 cells were treated with vehicle, P4 (10 μM), ML-SA1 (20 μM), or ML-SI1 (10 μM) as indicated for 8 hours and then infected with PRV HN1201 (MOI = 0.1) for 24 hours. p-DNM1, DNM1, and PRV gE were detected by immunoblotting. (L) TRPML1 in Mcoln1+/+ and Mcoln1-/- MEF cells was detected by immunoblotting. Data are expressed as the mean ± SD of 3 independent experiments. P-values were determined by Student’s t-test. P (TIF)