plhcx flag mpkm1  (Addgene inc)

Journal: eLife

Article Title: Glycolytic reliance promotes anabolism in photoreceptors

doi: 10.7554/elife.25946

Figure Lengend Snippet: Figure 3. PKM1 and PKM2 isoforms have nonequivalent roles. (A) Biased expression of M1 and M2 isoforms in retinal layers detected by IHC and ISH. (B) Immunoblot of retinal lysates from postnatal retina at different developmental stages. HEK293T cell lysates that were from untransfected (UT) cells, or those transfected with CAG-FLAGmuPKM1 (M1) or CAG-FLAGmuPKM2 (M2) as controls. Postnatal age in days. A, mature retina (P25–P30). (C) Outer segment phenotype of P45 mice after electroporation with constructs encoding mouse PKM2-specific shRNA (PKM2sh) and adding either mouse PKM1 (muPKM1) or human PKM2 (huPKM2). Selected areas in yellow boxes are magnified on the right. (D) Quantification of IS+OS lengths obtained in (C). n = 32–53 cells from 3 to 4 retinae. (E) Outer segment phenotype of dark-reared P31 mice electroporated with PKM2sh-encoding plasmid. The yellow- boxed region is magnified and presented on the right. (F) Quantification of IS+OS lengths obtained in (e). n = 75 cells from three retinae. (G) Secreted lactate from freshly isolated retinae from Pkm2fl/fl (fl/fl) (n = 12) or Rod-cre> Pkm2fl/fl (m2-/-) (n = 16) mice. (H) Outer segment phenotype after CAG promoter-driven overexpression of Flag-tagged mouse PKM1 or PKM2. Inset, higher magnification of IS and OS. (I) Quantification of IS+OS lengths obtained in (H). n = 35 cells from three retinae in PKM1 and PKM2 groups. ONL, outer nuclear layer. Data, Mean±SD. Statistics, one-way ANOVA with Tukey’s correction for panels D, I; two-way ANOVA with Tukey’s multiple comparison test for panel F; unpaired, two-tailed t-test with Kolmogorov- Smirnov correction for panel G. DOI: 10.7554/eLife.25946.012 The following source data and figure supplements are available for figure 3:

Article Snippet: The murine FLAG-tagged PKM1 and PKM2 cDNAs were obtained from Addgene (#44240 and #42512) and subcloned in pCAG-EN.

Techniques: Expressing, Western Blot, Transfection, Electroporation, Construct, shRNA, Plasmid Preparation, Isolation, Over Expression, Comparison, Two Tailed Test

Journal: eLife

Article Title: Glycolytic reliance promotes anabolism in photoreceptors

doi: 10.7554/elife.25946

Figure Lengend Snippet: Figure 4. FGF signaling regulates aerobic glycolysis and anabolism. (A) Schematic of PKM1 and PKM2 polypeptide showing Y105 is a shared epitope between PKM1 and PKM2. (B) Immunoprecipitation (IP) of PKM2 from adult retina followed by immunoblot (IB) for either PKM1, PKM2 or pY105 PKM. IP using isotype-matched antibody (IgG) is used alongside to control for nonspecific binding. Lysates from skeletal muscle (expresses PKM1) and 293T (expresses only PKM2) included as controls. Molecular weight marker positions are depicted on the right-hand-side (C) Retinal lysates were prepared from eyes harvested at 3-hr interval during the 12 hr light 12 hr dark cycle. T0 is the time point of light on in the room. The lysates were subjected to immunoprecipitation with anti-PKM2. Immunoprecipitates were probed for phosphorylation at Y105 by immunoblotting with the phospho-specific antibody. (D) Lysates from explants treated with candidate tyrosine kinase pathway inhibitors or vehicle control (DMSO) were subjected to immunoprecipitation with anti-PKM2. Immunoprecipitates were probed for phosphorylation at Y105 by immunoblotting with the phospho-specific antibody. (E) FGF inhibitors also reduce phosphorylation of LDHA at the Y10 residue. Phosphorylation of FRS2, an FGFR-interacting protein was included as a control. SDHA served as loading control. (F) Rate of lactate production from explants treated with DMSO (n = 5) or FGF inhibitors PD173074 (5 mM) (n = 6), PD173074 (20 mM) (n = 5), TKI258 (n = 6). (G) Steady-state ATP levels per retina in explants after culture with TKI258 or DMSO. The retinae were transferred to Krebs’-Ringer’s with NaN3 or NaCl (untreated group) for 30 min followed by harvest for ATP extraction. n = 7, DMSO+NaCl; n = 9, TKI258+NaCl; n = 9, DMSO+NaN3; n = 9, TKI258+NaN3. Data are Mean±SD. Statistics, Two-way ANOVA with Tukey’s correction. (H) NADPH steady state levels in explants as a percentage of those measured in freshly isolated retina. Explants were treated with DMSO, oxamate, PD173074, TKI258 or left untreated in culture medium. n = 4 groups. Unpaired t-test with Kolmogorov-Smirnov correction for indicated pairs. (I) NADP steady-state levels in explants as a percentage of those measured in freshly isolated retina. Explants were treated with DMSO, oxamate, PD173074, TKI258 or left untreated in culture medium. Oxamate, n = 5; rest, n = 6 groups. Unpaired t-test with Kolmogorov-Smirnov correction for indicated pairs. (J) Blocking glycolysis or FGF signaling reduced EU incorporation in nascent RNA. Explants were treated with DMSO, oxamate, TKI258 or Actinomycin D (RNA Pol II inhibitor) followed by incubation with EU. (K) Quantitative PCR analysis of transcripts to ascertain relative expression of FGF or non-FGF targets (Arr3, Rs1) in explants cultured with or without RPE/Sclera complex (+RPE or –RPE respectively). (L) Ability to produce lactate from neural retina increased when cultured in the presence of RPE/Sclera complex (+RPE) (n = 11) as compared to those that were cultured without the complex (-RPE) (n = 9). Addition of FGF2 in –RPE cultures restored the ability (-RPE+FGF2) (n = 8). Retinal explants were cultured with RPE attached in the explant culture medium. Before transferring them to Krebs’s-Ringer’s for lactate estimation, the RPE/Sclera complex was removed and intact neural retina was used. For –RPE conditions, neural retina was cultured in explant medium followed by transfer to Krebs’-Ringer’s. FGF2 was added to the explant culture medium but was absent in the Krebs’-Ringer’s for -RPE+FGF2 condition. Data depict median in 1–99 percentile box and whiskers plot. Hinges extend between 25th to 75th percentiles. Statistics, Ordinary one-way ANOVA with Tukey’s correction. ONL, outer nuclear layer. INL, inner nuclear layer. GCL, Ganglion cell layer. DOI: 10.7554/eLife.25946.020 The following source data and figure supplement are available for figure 4:

Article Snippet: The murine FLAG-tagged PKM1 and PKM2 cDNAs were obtained from Addgene (#44240 and #42512) and subcloned in pCAG-EN.

Techniques: Immunoprecipitation, Western Blot, Control, Binding Assay, Molecular Weight, Marker, Phospho-proteomics, Residue, Extraction, Isolation, Blocking Assay, Incubation, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Transferring