Journal: Scientific Reports

Article Title: Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway

doi: 10.1038/s41598-017-03477-3

Figure Lengend Snippet: AMPK activation is required for OMS-induced phagosomal maturation and antimicrobial responses. ( a and b ) BMDMs were treated with OMS-A (10 μM; for a ) or OMS-B (10 μM; for b ) for indicated times (0–18 h). The cell lysates were subjected to immunoblot analysis of p-AMPKα, p-ACC, and Actin. The entire blots are shown in Supplementary Fig. . The densitometric values for p-AMPKα were normalized to Actin ( bottom ). ( c and d ) BMDMs were transduced with non-specific shRNA (sh NS ) or Ampk -specific shRNA (sh Ampk )-expressing lentivirus for 48 h and then infected with ERFP-Mtb (moi = 10) for 4 h, followed by treatment with OMS-A or OMS-B for 24 h. ( c and d ) Mtb-ERFP (red), Alexa 488-conjugated-LC3 (green), and DAPI (blue) were detected by confocal analysis. Scale bar, 5 µm. ( c ) Representative confocal microscopic images from three independent samples are shown. ( d ) Quantitative analysis of cells showing the colocalization between LC3 and Mtb-ERFP. For each experiment, at least 100 cells were scored from 6 random fields. ( e ) After 3 days of infection, intracellular bacterial loads were determined by CFU assay. (inset) RT-PCR analysis of Ampk mRNA expression of transduction efficiency. Data shown are from one representative of at least three independent experiments (means ± SD of triplicates [ a , b bottom ; d ] samples]). *p < 0.05, ***p < 0.001, compared with sh NS . U, uninfected/untreated; SC, solvent control.

Article Snippet: The pLKO.1-based target shRNA plasmids for m Ampkα (Santa Cruz Biotechnology, sc-29674-SH) or h ATG5 (Santa Cruz Biotechnology, sc-41445-SH) were cotransfected into HEK293T cells using Lipofectamine 2000 (Invitrogen, 12566014, Carlsbad, CA, USA) for 72 h. Then, the lentivirus-containing supernatant was collected, filtered, and titrated.

Techniques: Activation Assay, Western Blot, Transduction, shRNA, Expressing, Infection, Colony-forming Unit Assay, Reverse Transcription Polymerase Chain Reaction, Solvent, Control

Journal: Scientific Reports

Article Title: Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway

doi: 10.1038/s41598-017-03477-3

Figure Lengend Snippet: OMS inhibits Mtb-induced inflammatory responses through AMPK activation. ( a ) BMDMs were infected with Mtb (moi = 10) for 4 h and then treated with OMS-A (1, 5, 10 μM) or OMS-B (1, 5, 10 μM) for 24 h. ( b ) BMDMs were transduced with adenoviral NF-ĸB-luciferase reporter plasmid for 36 h, and infected with Mtb and then treated with OMS-A or OMS-B for 6 h. The cells were harvested and NF-ĸB luciferase reporter activity was determined. ( c ) BMDMs were transduced with non-specific shRNA (sh NS ) or Ampk -specific shRNA (sh Ampk )-expressing lentivirus for 48 h and then infected with Mtb, followed by treatment with OMS-A or OMS-B for 24 h. ( a and c ) The supernatants were harvested and subjected of ELISA analysis of TNF-α, IL-6, IL-1β, and IL-12p40 production. All data represent the means ± SD of triplicates from each sample. **p < 0.01, ***p < 0.001, compared with SC ( a – c ). U, uninfected/untreated; SC, solvent control; ns, no significant.

Article Snippet: The pLKO.1-based target shRNA plasmids for m Ampkα (Santa Cruz Biotechnology, sc-29674-SH) or h ATG5 (Santa Cruz Biotechnology, sc-41445-SH) were cotransfected into HEK293T cells using Lipofectamine 2000 (Invitrogen, 12566014, Carlsbad, CA, USA) for 72 h. Then, the lentivirus-containing supernatant was collected, filtered, and titrated.

Techniques: Activation Assay, Infection, Transduction, Luciferase, Plasmid Preparation, Activity Assay, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Solvent, Control