Fig 1: Calpain 1 is required for PRRSV uncoating. (A) Co-localization of calpain 1 (Abcam, ab108400) and PRRSV-N. WT cells were fixed after CHR6 infection for 30 min, and subjected to fluorescence confocal experiments to detect calpain 1 (Green) and PRRSV-N (Red). Scale bar represents 20 µm. (B) Calpain 1 is required for events occurring before virus internalization. WT cells were infected with CHR6 for 2 h and then treated with calpain 1 inhibitor calpeptin (110 µM) for 24 h or pretreated with calpeptin for 2 h prior to virus infection for 24 h. Expression of PRRSV N protein was examined by western blotting to confirm the inhibitory effect of calpeptin on PRRSV replication. (C) An immunofluorescence-based assay for viral location in infected WT cells treated with DMSO as a negative control (NC) or calpeptin (calp., 110 µM) for 2 h at 37°C. Red fluorescence indicates virus and blue fluorescence indicates nucleus (DAPI). Scale bar represents 20 µm. (D,E) Viral attachment assay was performed in WT cells exposed to CHR6 (MOI = 1) for 1 h at 4°C in the presence of DMSO (NC) or calpeptin (110 µM) (D), and viral internalization and replication assays were performed in cells infected with CHR6 (MOI = 1) for 30 min (viral internalization phase), 5 h, or 10 h at 37°C in the presence of DMSO (NC) or calpeptin (110 µM) (E). Green fluorescence indicates absorbed virus, internalized virus or replicated virus, and blue fluorescence indicates nucleus (DAPI). Scale bar represents 20 µm. (F,G) Colocalization between CHR6 virions and the markers of early endosomes (EEA1) (F) and late endosomes (CI-M6PR) (G). Calpeptin-treated or DMSO-treated cells were inoculated with CHR6 strain (MOI = 1) for 1 h at 4°C and subsequently incubated for 30 min (F) or 45 min (G) at 37°C. Green fluorescence indicates virus located in cells, red fluorescence indicates early endosomes compartment (EEA1) or late endosomes (M6PR) and blue fluorescence indicates nucleus (DAPI). Yellow indicates colocalization (arrowheads) between EEA1 or M6PR with PRRSV-N. Scale bar represents 20 µm.
Fig 2: miR-124 inhibition increases Capn1 and CTNNB1 expression the lnc-NEAT1-downregulated MMC DN cell model. The MMC DN cell model was transfected with either lnc-KD + miR-124-inhibitor or lnc-KD + NC-inhibitor. (A) Capn1 and (B) CTNNB1 mRNA expression was measured by reverse transcription-quantitative PCR. (C) Capn1 and CTNNB1 protein expression was measured by western blotting, (D) which was quantified. miR, microRNA; Capn1, calpain 1; CTNNB1, catenin ß1; lnc-NEAT1, long non-coding RNA nuclear enriched abundant transcript 1; MMC, mouse mesangial cell; DN, diabetic nephropathy; lnc-KD, lnc-NEAT1 knockdown; NC-KD, negative control knockdown.
Fig 3: Lnc-NEAT1 expression, cell viability and apoptosis in lnc-NEAT1-downregulated MMC DN cells. (A-F) MMC cells were treated with either HG or NG. (A) Cell viability was measured using CCK-8 assay. (B) Capn1 and (C) CTNNB1 mRNA expression was measured by RT-qPCR. (D) Capn1 and CTNNB1 protein expression was measured by western blotting, (E) which was quantified. (F) Lnc-NEAT1 expression was measured in HG cells by RT-qPCR. (G-L) MMC cells were transfected with either lnc-KD or NC-KD plasmids after HG treatment. (G) Lnc-NEAT1 expression was measured by RT-qPCR after Lnc-NEAT1 knockdown. (H) Cell viability was measured using CCK-8 assay. (I) Cell apoptosis was measured using flow cytometry, (J) which was quantified. (K) Protein expression levels of cleaved caspase 3 and cleaved PARP in the lnc-KD and NC-KD groups were measured by western blotting, (L) which was quantified. Lnc-NEAT1, long non-coding RNA nuclear enriched abundant transcript 1; NG cells, normal glucose-treated cells; HG cells, high glucose-treated cells; lnc-KD, lnc-NEAT1 knockdown; NC-KD, negative control knockdown; MMC, mouse mesangial cells; DN, diabetic nephropathy; RT-qPCR, reverse transcription-quantitative PCR; CCK-8, Cell Counting Kit-8; OD, optical density; OC, osmotic control; capn1, calpain 1; CTNNB1, ß-catenin; PARP, poly-ADP ribose polymerase.
Supplier Page from Abcam for Anti-Calpain 1 antibody [EPR3319]