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

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

Journal: eLife

Article Title: Glycolytic reliance promotes anabolism in photoreceptors

doi: 10.7554/eLife.25946

Figure Lengend Snippet: ( 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 Pkm2 fl/fl (fl/fl) (n = 12) or Rod-cre> Pkm2 fl/f l (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: http://dx.doi.org/10.7554/eLife.25946.012 10.7554/eLife.25946.013 Figure 3—source data 1. Source data for . DOI: http://dx.doi.org/10.7554/eLife.25946.013

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: Retinal cross section of a 6-week-old Rod-cre; Pkm2 fl/fl (depicted previously in ) stained for PKM1, Rhodopsin (RHO) and counterstained with DAPI. The region of slightly longer outer segments is demarcated with arrows. DOI: http://dx.doi.org/10.7554/eLife.25946.018

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

Techniques: Staining

Journal: eLife

Article Title: Glycolytic reliance promotes anabolism in photoreceptors

doi: 10.7554/eLife.25946

Figure Lengend Snippet: Retinal cross-section of a 37-week-old Rod-cre; PKM2 fl/fl mouse stained for PKM2, Rhodopsin (RHO) and counterstained with DAPI. Slight nuclear disorganization in the retinal mosaic corresponding to PKM2 loss is demarcated in the panel corresponding to the DAPI channel (lower left). Aberrant mislocalized RHO + cell is marked by arrow. DOI: http://dx.doi.org/10.7554/eLife.25946.019

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

Techniques: Staining

Journal: eLife

Article Title: Glycolytic reliance promotes anabolism in photoreceptors

doi: 10.7554/eLife.25946

Figure Lengend Snippet: ( A ) Schematic of PKM1 and PKM2 polypeptide showing Y 105 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 pY 105 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. T 0 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 Y 105 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 Y 105 by immunoblotting with the phospho-specific antibody. ( E ) FGF inhibitors also reduce phosphorylation of LDHA at the Y 10 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 NaN 3 or NaCl (untreated group) for 30 min followed by harvest for ATP extraction. n = 7, DMSO+NaCl; n = 9, TKI258+NaCl; n = 9, DMSO+NaN 3 ; n = 9, TKI258+NaN 3 . 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: http://dx.doi.org/10.7554/eLife.25946.020 10.7554/eLife.25946.021 Figure 4—source data 1. Source data for . DOI: http://dx.doi.org/10.7554/eLife.25946.021

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

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Gene set enrichment analysis of RNA-seq data of RPE1 TP53 KO control and EIF2A KO cells (more detailed analysis in Suppl. Fig. 3A). Circle size represents gene set size. (B, C) SILAC-based proteomics analysis of RPE1 TP53 KO control and EIF2A KO cells, showing differential protein expression at baseline (panel B) and upon AZD1775 treatment for 6, 24 and 48 h (panel C). (D) RPE1 TP53 KO control and EIF2A KO cells were treated with AZD1775 or Debio 0123 (1 µM) for 24 h, followed by a 10’ puromycin pulse. Puromycin incorporation was visualized by immunoblotting (left panel). Quantification of immunoblots is indicated (right panel). Data represent mean ± SD; RPE1 TP53 KO EIF2A KO treated with Debio 0123 (n=4); control RPE1 TP53 KO treated with DMSO or AZD1775 (n=7); Other conditions (n=5). (E) RPE1 TP53 KO PAC KO cells were treated with AZD1775 (250 nM) and ISRIB for 24 h and subsequently labeled with L-azidohomoalanine (AHA). Immunoblot of AHA-labeled proteins and quantification is shown. Data represent mean ± SD (n=2). (F) Schematic overview of the CRISPR/Cas9 genome-wide AZD1775 resistance screen. (G) MAGeCK scores for individual genes in the RPE1 TP53 KO AZD1775 positive selection screen (day 12 vs day 0). (H) Cell confluency analysis of parental RPE1 TP53 KO PAC KO or GCN2 KO cells. Mean ± SD, n=3 for control and GCN2 KO #1, n=2 for GCN2 KO #2. (I) Quantification of clonogenic survival assays of parental RPE1 TP53 KO PAC KO and ATF4 KO #1 and #2 cells (corresponding images provided in Suppl. Fig. 3H). Mean ± SD, n=3 for all conditions. (J, K) Representative histograms (panel J) and quantification (panel K) of ATF4-mScarlet flow cytometry measurements in RPE1 TP53 KO ATF4-mScarlet reporter cells treated with thapsigargin (1 µM), AZD1775 (1 µM), Debio 0123 (1 µM) and/or A92 (1 µM) for 24 h. Data represent mean ± SD (n=3). (L) Parental RPE1 TP53 KO PAC KO , ATF4 KO #1 and GCN2 KO #2 cells were treated with AZD1775 (0, 250 or 500 nM) for 24 h, and indicated proteins were immunoblotted. Statistical analysis of panels D, K: unpaired t-test with p ≤ 0.05 considered significant.

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: RNA Sequencing, Control, Expressing, Western Blot, Labeling, CRISPR, Genome Wide, Selection, Flow Cytometry

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) RPE1 TP53 KO PAC KO cells were treated with AZD1775 (250 nM) for 6 h in the presence or absence of ISRIB (1 μM), pulse labeled with EdU, and processed for quantitative image-based cytometry. Cell cycle stage was defined by DNA content and EdU positivity. Median ATF4 intensity from n=2 experiments is shown. (B, C) Experimental set-up (panel B). RPE1 TP53 KO ATF4-mScarlet-NLS reporter cells were treated overnight with nocodazole. Mitotic cells were isolated and replated in the presence or absence of palbociclib. After 2 h AZD1775 (1 µM) and/or A92 (1 µM) was added for 20 h (Panel B). mScarlet levels in RPE1 TP53 KO ATF4-mScarlet reporter cells were measured by flow cytometry. Data represent mean ± SD (n=3)(panel C). (D, E) Flow cytometry gating strategy (panel D) and analysis of γH2AX in RPE1 TP53 KO cells after treatment with AZD1775 (1 µM) and/or A92 (1 µM) in the absence (panel E, left) or presence (panel E, right) of nocodazole. Data represent mean ± SD (n=4). (F) Flow cytometry analysis of MPM2-positivity in RPE1 TP53 KO cells after treatment with nocodazole, doxorubicin, AZD1775 (1 µM) and/or A92 (1 µM). Data represent mean ± SD (n=3). (G) Flow cytometry analysis of MPM2-positivity in control RPE1 TP53 KO PAC KO and ATF4 KO #2 or GCN2 KO #2 cells after treatment with nocodazole, doxorubicin and AZD1775 (1 µM). Data represent mean ± SD (n=3). (H) Immunoblot of resting PBMCs or PBMCs stimulated with anti-CD3/CD28 beads. PBMCs were treated with DMSO, AZD1775 (500 nM) or thapsigargin (500 nM) for 24 h. (I) RPE1 TP53 KO PAC KO and ATF4 KO #2 or GCN2 KO #2 cells were treated with AZD1775 and/or cisplatin for 5 days. Cell survival was analyzed by MTT conversion. Data represent mean ± SD (n=4). Statistical analysis was performed using unpaired t-tests, with p ≤ 0.05 considered significant.

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: Labeling, Cytometry, Isolation, Flow Cytometry, Control, Western Blot

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Relative gene and protein expression upon treatment with AZD1775 (24 h, 1 µM) measured by Ribo-seq and RNA-seq. (B) Differential protein expression upon treatment with AZD1775 (24 h, 1 µM) measured by Ribo-seq. (C) Pathway enrichment analysis of Ribo-seq data shown in (M). (D) Gene expression of ATF4-target genes DDIT4, DDIT3, ATF3 and PPP1R15A upon treatment with AZD1775 (24 h, 1 µM). (E) Codon usage in RPE1 TP53 KO cells upon treatment with DMSO or AZD1775 (24 h, 1 µM).

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Relative ATF4-mScarlet MFI in RPE1 ATF4-mScarlet reporter cells after treatment with 125, 250, 500, or 1000 nM of AZD1775, RP-6306, VE-822 or AZD7762. Data represent mean ± SD (DMSO: n=4; other conditions: n=3). Statistical analysis was performed using a one-way ANOVA multiple comparisons with p≤0.05 considered significant. (B) Immunoblot of RPE1 TP53 KO cells treated with siRNA targeting WEE1 for 72 h and AZD1775 (250 nM) for 2.5 or 5 h. (C, D) NanoBRET kinase target engagement assay performed with HEK293T cells transfected with WEE1-NanoLuc (panel C) and NanoLuc-GCN2 (panel D) and incubated with K-10 tracer (0.5 μM) and indicated doses of CC1, AZD1775, Debio 0123 and ZNL-02-096 for 2 h prior to substrate addition and BRET signal detection.

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: Western Blot, Transfection, Incubation

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Relative ATF4-mScarlet MFI in RPE1 TP53 KO ATF4-mScarlet reporter cells after treatment with the indicated doses of Debio 0123. Data represent mean ± SD (n=3). (B) Cell viability in parental RPE1 TP53 KO PAC KO , GCN2 KO #1 and GCN2 KO #2 cells after treatment with Debio 0123 and RP-6306 at the indicated doses (n=5). (C) ZIP synergy scores from cell survival matrices shown in and . Data represent mean ± SD (n=5). (D) Synergy plots with ZIP synergy scores of heatmaps shown in . (E) Relative ATF4-mScarlet MFI in RPE1 TP53 KO ATF4-mScarlet reporter cells treated with the indicated doses of AZD1775, Debio 0123 and RP-6306. Data represent mean ± SD (n=4).

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: