Journal: Cell metabolism

Article Title: Targeting Peripheral CB 1 Receptors Reduces Ethanol Intake via a Gut-Brain Axis

doi: 10.1016/j.cmet.2019.04.012

Figure Lengend Snippet: (A) Time course and rate of increase in oleate-driven respiratory activity in MGN3–1 cells. Cells were treated with 10 or 100 nM JD5037, 2.5 M of FCCP (positive control) or vehicle in the presence of the fluorescent extracellular O2 consumption reagent. Oxidative respiration was recorded every 90 sec and the slope of the initial linear increase was calculated. Points and bars are means ± SEM from n = 11–15 experiments, as indicated. *P < 0.05 compared to vehicle. (B) Verification of Cnr1 knockdown by rtPCR in cells used in panel C. Cellular uptake of the constructs was verified by fluorescent microscopy (20x magnification) and the degree of knockdown was determined by rt-PCR, *P < 0.05, n = 4. (C) Oleate-driven oxidative activity in MGN3–1 cells with shRNA-mediated knockdown of Cnr1 expression and their mock-transfected controls, n = 3–8 experiments, as indicated, *P < 0.05.

Article Snippet: Scramble shRNA with GFP Adenovirus (Ad-scramble-shRNA) , Vector Biolabs , 1122.

Techniques: Activity Assay, Positive Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Construct, Microscopy, shRNA, Expressing, Transfection

Journal: eLife

Article Title: Glycolytic reliance promotes anabolism in photoreceptors

doi: 10.7554/eLife.25946

Figure Lengend Snippet: ( A ) Schematic of the PKM genomic locus and depiction of generation of M1 and M2 isoforms by alternative splicing. ( B ) Plasmids encoding cDNA of either mouse PKM1 or PKM2 and destabilized GFP (dGFP) were transfected in 293T cells. CAG-mCherry served as transfection control. Plasmids encoding candidate shRNAs driven by the U6 promoter were cotransfected. ( C ) Live imaging of dGFP or mCherry in transfected cells. PKM1 +2 sh encodes for shRNA that targets both M1 and M2, whereas the PKM2sh codes for shRNA that is M2-specific. Scale bar, 50 μm. ( D ) Effect of shRNAs on protein steady state. Representative immunoblot of 293T cells transfected with Flag-tagged muPKM1 or muPKM2 driven by the CAG promoter and co-transfected with a plasmid encoding for mCherry and indicated shRNA-encoding constructs. Forty-eight hour post-transfection, cells were lysed and lystaed used for immunoblots using indicated antibodies on the left. This panel also represents shRNAs that had a knockdown effect in the screen in (b), but were not considered in favor of PKM1 +2 owing to its strong knockdown effect. UT, untransfected 293T (endogenously express PKM2 but not PKM1). ( E ), Representative immunoblot of 293 T cells transfected with Flag-tagged muPKM1 or muPKM2 driven by the CAG promoter and cotransfected with PKM2sh from (c) (‘+’ lanes) or empty sh vector (‘- ‘lanes) and harvested 24 hr later for lysate preparation. ( F ) Human and mouse M2 exon alignment. The region targeted by PKM2sh is highlighted. This shRNA did not knockdown the human PKM2. ( G ) In vivo electroporation of a plasmid encoding PKM1 +2 shRNA resulted in photoreceptors with significantly shorter inner plus outer segments (top left). This phenotype could be rescued by coelectroporation of a construct encoding human PKM2 cDNA (top right and bottom left). In 4/6 retinae (top right), many photoreceptors lacked clear borders distinguishing inner and outer segments (arrows) while some photoreceptors looked normal (arrowheads). In 2/6 retinae (bottom left), the morphology resembled that of control retinae. ( H ) Retinal cross section of a 6 week old Rod-cre; PKM2 fl/fl mouse stained for PKM1. ( I ) Retinal cross section of a P40 mouse electroporated with PKM2sh and CAG-mGFP. Arrows mark inner segments of electroporated photoreceptors. ( J ) Representative immunoblot of 293T cells transfected with Flag-tagged muPKM1 or muPKM2 driven by the CAG promoter. GAPDH served as loading control. DOI: http://dx.doi.org/10.7554/eLife.25946.015

Article Snippet: Designed sh oligos were subcloned in pLKO.1 TRC backbone to be driven by the U6 promoter (Addgene, Cambridge, MA, #10878) and the sequences used in this manuscript are listed in .

Techniques: Alternative Splicing, Transfection, Control, Imaging, shRNA, Western Blot, Plasmid Preparation, Construct, Knockdown, In Vivo, Electroporation, Staining

Journal: Cell reports

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis

doi: 10.1016/j.celrep.2021.110008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Briefly, a guide RNA (5′-GCAGATGTAGTGTTTCCACA-3′) targeting the open reading frame in the immediate vicinity of the stop codon was cloned into the Cas9 expression vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (gift from Feng Zhang; ), Addgene plasmid #42230).

Techniques: Derivative Assay, Recombinant, Electron Microscopy, Transfection, Expressing, Plasmid Preparation, Software, Cell Analysis, Gas Chromatography, Mass Spectrometry