Journal: iScience

Article Title: PIMT regulates hepatic gluconeogenesis in mice

doi: 10.1016/j.isci.2023.106120

Figure Lengend Snippet: PIMT is phosphorylated by PKA at Ser 656 (A) Sepharose bound GST fused PIMT-C was subjected to kinase reaction in the presence of HeLa nuclear lysate, constitutively active purified MAPKS (ERK1 and ERK2), or PKA. (B) Primary Hepatocytes infected with lentivirus expressing PIMT-V5 were exposed to Forskolin (10 μM) with or without H89 (20 μM) or RP (20 μM) for 1h and then subjected to IP with PKA substrate antibody followed by immunoblot with indicated antibodies. (C) Densitometric quantification of Figure 4 B. The phosphorylation signals were normalized with their corresponding input. Numerical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. ∗∗∗∗p < 0.001 compared to DMSO treated cells, a p< 0.001 compared to Forskolin treated cells. (D) Mice were infected with lentivirus expressing PIMT-V5 (n = 3). Post 7 days of infection, mice were fasted for 8h and liver lysates were subjected to IP with PKA substrate antibody followed by immunoblots with anti-V5. (E) Densitometric quantification of Figure 4 D. The phosphorylation signals were normalized with the corresponding input signals. Numerical data are expressed as mean ± SD. Statistical analysis was performed using unpaired Student’s t -test (two-tailed) ∗∗∗ p < 0.005, versus the corresponding input. (F) Glutathione Sepharose beads bound GST-PIMT-C (W and mutants) were subjected to kinase assay with active and purified PKA. Double mutations at the PKA recognition site (RxxS, Ser 656 , and Ser 851 ) abolished the phosphorylation of PIMT. (G) Primary hepatocytes isolated from female mice infected with either PIMT (W) or PIMT S656A were treated with Forskolin for 4h and then subjected to IP with PKA substrate antibody followed by immunoblots with the defined antibodies (n = 4). (H) Densitometric quantification of Figure 4 G. The phosphorylation signals were normalized with the corresponding input signals. Numerical data are expressed as mean ± SD. Statistical analysis was performed using unpaired Student’s t -test (two-tailed) ∗∗∗ p < 0.005, versus the corresponding input. (I) C57BL/6 male mice were tail-vein injected with lentivirus expressing PIMT (wt or S656A) (n = 3). Post 7 days of injection, mice fasted for 8h. Liver lysates were subjected to IP with PKA substrate antibody followed by immunoblots with the defined antibodies. (J) Densitometric quantification of Figure 4 G. The phosphorylation signals were normalized with the corresponding input signals. Numerical data are expressed as mean ± SD. Statistical analysis was performed using unpaired Student’s t-test (two-tailed) ∗∗∗ p < 0.005, versus the corresponding input. (K) 8h fasted liver lysates subjected to IP with PKA substrate antibody followed by immunoblots with the defined antibodies (n = 3). (L) Densitometric quantification of Figure 4 I. The phosphorylation signals were normalized with the corresponding input signals. Statistical analysis was performed using unpaired Student’s t-test (two-tailed) ∗∗ p < 0.01, versus the corresponding input. (M) HepG2 cells were transfected with the pGL3-PEPCK promoter and PIMT (W or mutants) encoding constructs with or without PKAc. Post-transfection cells were lysed, and luciferase readout was measured. The values were normalized with corresponding Renilla luciferase activity and expressed relative to PEPCK-Luc (unphosphorylated) (column 1), set to 1. Data are representative of 5 independent experiments and expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. d p< 0.001 vs PECK-Luc (unphosphorylated) ∗∗∗∗ p < 0.001 compared to PEPCK-Luc with PKAc transfected cells. γ p< 0.05, α p < 0.005 compared to PEPCK-Luc + PIMT (W) without PKAc, D p< 0.001 compared to PEPCK-Luc + PIMT (W) + PKAc, PIMT-W: PIMT wild type, PIMT-A: PIMT S656A mutant, PIMT-D: PIMT S656D mutant.

Article Snippet: Recombinant Human Active ERK2 Protein , RnD Systems , 1230-KS-010.

Techniques: Purification, Infection, Expressing, Western Blot, Phospho-proteomics, Two Tailed Test, Kinase Assay, Isolation, Injection, Transfection, Construct, Luciferase, Activity Assay, Mutagenesis

Journal: iScience

Article Title: PIMT regulates hepatic gluconeogenesis in mice

doi: 10.1016/j.isci.2023.106120

Figure Lengend Snippet:

Article Snippet: Recombinant Human Active ERK2 Protein , RnD Systems , 1230-KS-010.

Techniques: Recombinant, SYBR Green Assay, Isolation, Reverse Transcription, Colorimetric Assay, Mutagenesis, PCR Cloning, Cloning, Software, Western Blot

Journal: Cell reports

Article Title: Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target.

doi: 10.1016/j.celrep.2022.110923

Figure Lengend Snippet: Figure 1. Human PRDX6 is internalized by P. falciparum along with hemoglobin during HCCU (A) Immunofluorescence microscopy shows even cytosolic staining of PRDX6 in uninfected RBCs. In early and late trophozoites, multiple PRDX6 foci (white arrowheads) were observed adjacent to the parasite FV. DNA, Hoechst 33342 (nuclei). Scale bars, 5 mm. Representative images from three independent exper- iments are shown. See also Figures S1C and S1D. (B–F) Immuno-transmission electron microscopy (TEM) using monoclonal mouse anti-PRDX6 antibody. (B) Uninfected RBCs showing even cytosolic staining for PRDX6. (C–F) P. falciparum-infected RBCs show co-localization of PRDX6 and hemoglobin in vesicles at the inner surface of the parasite plasma membrane in structures that resemble cytostomes (black arrows), at the FV (white arrows), or within the parasite cytosol (striped gray arrow). Scale bars, 500 nm. Representative images from three independent experiments are shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti human PRDX6 (1A11) Santa Cruz Biotechnology Cat# sc-59671; RRID: AB_632188 Goat anti human Hemoglobin HRP-coupled Bethyl Cat# A80-134P; RRID: AB_67041 Rat anti mouse CD71 APC Thermo Fisher Scientific Cat# 17-0711-80; RRID: AB_1834356 Rabbit anti-PfNAPL Singh et al., 2010 N/A Goat anti mouse HRP-coupled Promega Cat# W4021; RRID: AB_430834 Goat anti rabbit HRP-coupled Promega Cat# W4011; RRID: AB_430833 Goat anti mouse Alexa Fluor 488-coupled Thermo Fisher Scientific Cat# A-11029; RRID: AB_2534088 Protein A gold 10nm UMC Utrecht Cat# PAG 10 nm 200mL Rabbit Anti-Mouse Immunoglobulins Dako/Agilent Cat# Z0259; RRID: AB_2532147 Bacterial and virus strains E. Coli BL21 (DE3) Sigma Cat# CMC0014 Chemicals, peptides, and recombinant proteins Darapladib SelleckChem Cat# S7520 ATK Cayman Cat# 62120 MAFP Enzo Cat# BML-ST360 Varespladib (LY3115920) Cayman Cat# 18267 P11 Cayman Cat# 17507 Alpha-Tocopherol Sigma Cat# T3251 Dihydroartemisinin Cayman Cat# 19846 E64 Sigma Cat# E3132 BODIPY 581/591 C11 Invitrogen Cat# D3861 LysoSensorTM Yellow/Blue dextran, 10,000 MW Invitrogen Cat# L22460 SYTO 61 Invitrogen Cat# S11343 SYBR Green I Lonza Cat# 50513 Hoechst 33342 Invitrogen Cat# H3570 TAMRA-FP ABPP Probe Invitrogen Cat# 88318 SYPRO Orange Invitrogen Cat# S6651 [14C]-DPPC American Radiolabeled Chemicals Cat# ARC0715-10UCI Cholesterol Sigma Cat# C8667 DPPC Avanti Lipids Cat# 850355P Egg-yolk PC Sigma Cat# P8318 Egg-yolk PG Sigma Cat# P3556 UltimaGold liquid scintillation cocktail Perkin Elmer Cat# 6013179 Saponin Sigma Cat# 47036 Mg-ATP Sigma Cat# A9187 Recombinant MAPK (ERK2) R&D Systems Cat# 1230-KS Recombinant PRDX6 This study N/A Recombinant PfGluPho Laboratory of Katja Becker, Gießen, Germany (Jortzik et al., 2011) N/A NADP+ Cayman Cat# 10004675 Glucose-6-phosphate Sigma Cat# G7879 Butylated hydroxytoluene (BHT) Sigma Cat# B1378 2-thiobarbituric acid (TBA) Sigma Cat# T5500 (Continued on next page) e1 Cell Reports 39, 110923, June 14, 2022

Techniques: Microscopy, Staining, Transmission Assay, Electron Microscopy, Infection, Clinical Proteomics, Membrane

Journal: Cell reports

Article Title: Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target.

doi: 10.1016/j.celrep.2022.110923

Figure Lengend Snippet: Figure 2. Darapladib selectively inhibits PRDX6 and blocks P. falciparum growth by impeding lipid-peroxidation repair (A) Binding of TAMRA-FP with recombinant PRDX6 in the presence of different PLA2 inhibitors in activity-based protein profiling (ABPP) assays. Reduced labeling of PRDX6 by TAMRA-FP identifies PLA2 inhibitors that bind PRDX6. See also Figure S2C. (B) Inhibition of P. falciparum ring-to-schizont progression and blood-stage growth with PLA2 inhibitors. Parasites were treated at ring stage, and growth to schizont stage in the same cycle (‘‘progression’’) or to next-generation rings (‘‘growth’’) was scored by flow cytometry using DNA intercalating fluorescent dye SYBR Green I. See also Table S1. (C) Representative light microscopy images of control and treated P. falciparum blood stages showing arrest at the trophozoite stage following treatment with different inhibitors at ring stage. Scale bars, 5 mm. (D) Radioactive PLA2 activity assay using phosphorylated recombinant human PRDX6 in presence or absence of Darapladib. Darapladib inhibits PLA2 activity of PRDX6 with IC50 of z0.5 mM. (E) Flow-cytometric measurement of in vivo growth curves of P. yoelii YM in C57BL/6 WT and prdx6/ mice. In vivo infection was performed two times inde- pendently with five mice per group. (F) Ex vivo ring-to-schizont progression of P. yoelii YM in CD71 mature RBCs from WT and prdx6/ mice in the presence and absence of Darapladib. Darapladib inhibits P. yoelii YM progression from ring-to-schizont stage in WT RBCs at a concentration of 1 mM but not in prdx6/ mouse RBCs. (G) ITDR-CETSA analysis of protein target engagement by Darapladib (0–100 mM) in trophozoite lysate. Distribution of protein stabilizations, under 50C (red circle), 55C (blue triangle), and 60C (green squares) thermal challenges, is plotted as a function of R2 value (goodness of curve fit) against DAUC (area under the curve of heat-challenged sample normalized against non-denaturing 37C control) for all proteins detected in the assay. Two and half times of median absolute

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti human PRDX6 (1A11) Santa Cruz Biotechnology Cat# sc-59671; RRID: AB_632188 Goat anti human Hemoglobin HRP-coupled Bethyl Cat# A80-134P; RRID: AB_67041 Rat anti mouse CD71 APC Thermo Fisher Scientific Cat# 17-0711-80; RRID: AB_1834356 Rabbit anti-PfNAPL Singh et al., 2010 N/A Goat anti mouse HRP-coupled Promega Cat# W4021; RRID: AB_430834 Goat anti rabbit HRP-coupled Promega Cat# W4011; RRID: AB_430833 Goat anti mouse Alexa Fluor 488-coupled Thermo Fisher Scientific Cat# A-11029; RRID: AB_2534088 Protein A gold 10nm UMC Utrecht Cat# PAG 10 nm 200mL Rabbit Anti-Mouse Immunoglobulins Dako/Agilent Cat# Z0259; RRID: AB_2532147 Bacterial and virus strains E. Coli BL21 (DE3) Sigma Cat# CMC0014 Chemicals, peptides, and recombinant proteins Darapladib SelleckChem Cat# S7520 ATK Cayman Cat# 62120 MAFP Enzo Cat# BML-ST360 Varespladib (LY3115920) Cayman Cat# 18267 P11 Cayman Cat# 17507 Alpha-Tocopherol Sigma Cat# T3251 Dihydroartemisinin Cayman Cat# 19846 E64 Sigma Cat# E3132 BODIPY 581/591 C11 Invitrogen Cat# D3861 LysoSensorTM Yellow/Blue dextran, 10,000 MW Invitrogen Cat# L22460 SYTO 61 Invitrogen Cat# S11343 SYBR Green I Lonza Cat# 50513 Hoechst 33342 Invitrogen Cat# H3570 TAMRA-FP ABPP Probe Invitrogen Cat# 88318 SYPRO Orange Invitrogen Cat# S6651 [14C]-DPPC American Radiolabeled Chemicals Cat# ARC0715-10UCI Cholesterol Sigma Cat# C8667 DPPC Avanti Lipids Cat# 850355P Egg-yolk PC Sigma Cat# P8318 Egg-yolk PG Sigma Cat# P3556 UltimaGold liquid scintillation cocktail Perkin Elmer Cat# 6013179 Saponin Sigma Cat# 47036 Mg-ATP Sigma Cat# A9187 Recombinant MAPK (ERK2) R&D Systems Cat# 1230-KS Recombinant PRDX6 This study N/A Recombinant PfGluPho Laboratory of Katja Becker, Gießen, Germany (Jortzik et al., 2011) N/A NADP+ Cayman Cat# 10004675 Glucose-6-phosphate Sigma Cat# G7879 Butylated hydroxytoluene (BHT) Sigma Cat# B1378 2-thiobarbituric acid (TBA) Sigma Cat# T5500 (Continued on next page) e1 Cell Reports 39, 110923, June 14, 2022

Techniques: Binding Assay, Recombinant, Activity Assay, Labeling, Inhibition, Cytometry, SYBR Green Assay, Light Microscopy, Control, In Vivo, Infection, Ex Vivo, Concentration Assay, Drug discovery

Journal: Cell reports

Article Title: Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target.

doi: 10.1016/j.celrep.2022.110923

Figure Lengend Snippet: Figure 3. Inhibition of PRDX6 blocks vesicular transport of hemoglobin-containing vesicles (HCVs) to the FV (A) Light microscopy of P. falciparum blood-stage cultures treated at the ring stage with Dar, E64, or Dar + E64 and incubated for 20 h. Treatment of P. falciparum rings with E64 resulted in bloating of the FV with undigested hemoglobin. In contrast, treatment with either Dar alone or with E64 prevented E64-mediated bloating of the FV, indicating a role of PRDX6 upstream of hemoglobin digestion. Scale bars, 5 mm. Representative images from three independent experiments are shown. (B) Quantification of FV size observed in (A). (C) TEM of P. falciparum blood-stage cultures from (A). Dar arrested transport of HCVs within the parasite cytosol. Scale bars, 500 nm. (D) Fluorescence microscopy. Use of pH-sensitive fluorescent probe to observe HCCU in P. falciparum blood stages following treatment with Dar. RBCs were preloaded with pH-sensitive LysoSensor Blue/Yellow, infected with P. falciparum, treated with Dar at the ring stage, and imaged at the trophozoite stage. Dar treatment prevented transport of RBC cytosol (neutral pH, blue) to the FV (acidic, yellow). Scale bars, 5 mm. DIC, differential interference contrast; LS435, LysoSensor at neutral pH; LS597, LysoSensor at acidic pH; SYTO61, DNA (nuclei). (E) Quantification of Yellow/Blue signal ratio within the FV observed by fluorescence microscopy in (D). (F) Flow-cytometric measurement of Yellow/Blue signal in P. falciparum-infected RBCs treated with Dar as described in (D). (A–F) Representative images or means ± SD from three independent experiments, unpaired t tests.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti human PRDX6 (1A11) Santa Cruz Biotechnology Cat# sc-59671; RRID: AB_632188 Goat anti human Hemoglobin HRP-coupled Bethyl Cat# A80-134P; RRID: AB_67041 Rat anti mouse CD71 APC Thermo Fisher Scientific Cat# 17-0711-80; RRID: AB_1834356 Rabbit anti-PfNAPL Singh et al., 2010 N/A Goat anti mouse HRP-coupled Promega Cat# W4021; RRID: AB_430834 Goat anti rabbit HRP-coupled Promega Cat# W4011; RRID: AB_430833 Goat anti mouse Alexa Fluor 488-coupled Thermo Fisher Scientific Cat# A-11029; RRID: AB_2534088 Protein A gold 10nm UMC Utrecht Cat# PAG 10 nm 200mL Rabbit Anti-Mouse Immunoglobulins Dako/Agilent Cat# Z0259; RRID: AB_2532147 Bacterial and virus strains E. Coli BL21 (DE3) Sigma Cat# CMC0014 Chemicals, peptides, and recombinant proteins Darapladib SelleckChem Cat# S7520 ATK Cayman Cat# 62120 MAFP Enzo Cat# BML-ST360 Varespladib (LY3115920) Cayman Cat# 18267 P11 Cayman Cat# 17507 Alpha-Tocopherol Sigma Cat# T3251 Dihydroartemisinin Cayman Cat# 19846 E64 Sigma Cat# E3132 BODIPY 581/591 C11 Invitrogen Cat# D3861 LysoSensorTM Yellow/Blue dextran, 10,000 MW Invitrogen Cat# L22460 SYTO 61 Invitrogen Cat# S11343 SYBR Green I Lonza Cat# 50513 Hoechst 33342 Invitrogen Cat# H3570 TAMRA-FP ABPP Probe Invitrogen Cat# 88318 SYPRO Orange Invitrogen Cat# S6651 [14C]-DPPC American Radiolabeled Chemicals Cat# ARC0715-10UCI Cholesterol Sigma Cat# C8667 DPPC Avanti Lipids Cat# 850355P Egg-yolk PC Sigma Cat# P8318 Egg-yolk PG Sigma Cat# P3556 UltimaGold liquid scintillation cocktail Perkin Elmer Cat# 6013179 Saponin Sigma Cat# 47036 Mg-ATP Sigma Cat# A9187 Recombinant MAPK (ERK2) R&D Systems Cat# 1230-KS Recombinant PRDX6 This study N/A Recombinant PfGluPho Laboratory of Katja Becker, Gießen, Germany (Jortzik et al., 2011) N/A NADP+ Cayman Cat# 10004675 Glucose-6-phosphate Sigma Cat# G7879 Butylated hydroxytoluene (BHT) Sigma Cat# B1378 2-thiobarbituric acid (TBA) Sigma Cat# T5500 (Continued on next page) e1 Cell Reports 39, 110923, June 14, 2022

Techniques: Inhibition, Light Microscopy, Incubation, Fluorescence, Microscopy, Infection