Journal: Autophagy

Article Title: GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.

doi: 10.1080/15548627.2021.1988357

Figure Lengend Snippet: Figure 5. GNS561 targets PPT1. (A) Nano differential scanning fluorimetry assays comparing GNS561 + PPT1 and HCQ + PPT1 against the apo-PPT1 ligand. Data represent the mean first derivative values (solid lines) ± SEM (shaded areas) of two experiments. SD from the mean is indicated by the light-color shading around the mean-line. Tm were determined by detecting the maximum of the first derivative of the fluorescence ratios. ΔTm values of each compound condition were determined by subtracting average Tm of PPT1 (in the respective buffer) by the average Tm of the respective compound condition. (B) PPT1 enzymatic activity of HepG2 cells treated with GNS561 for 3 h. HCQ and HDSF were used as positive controls. The results were compared to the diluent of GNS561 (control condition). (C) Representative immunoblotting of LC3-II in HepG2 cells treated with GNS561 for 16 h in the presence or absence of NtBuHA (8 mM). GAPDH was used as a loading control. Fold changes of normalized LC3-II level were calculated against the control condition (diluent of GNS561 + diluent of NtBuHA). (D) Cell viability percent against the control condition (diluent of GNS561 + diluent of NtBuHA) after 24 h of treatment with GNS561 in the presence or absence of NtBuHA (8 mM). (E) Fold change of normalized LC3-II (norm LC3-II) level were calculated against the control condition (diluent of GNS561) in HepG2 cells WT or siRNA-PPT1 treated by GNS561 for 24 h. GAPDH was used as a loading control. (F) Ratio of norm LC3-II between siRNA-PPT1 and WT HepG2 cells treated by GNS561 for 24 h. (G) Cell viability percent against the control condition (diluent of GNS561) after 24 h of treatment with GNS561 of WT and siRNA-PPT1 HepG2 cells. (H) Staining of lysosomes (LAMP2, green), MTOR (red) and nucleus (4′,6-diamidino-2-phenylindole [DAPI], blue) after treatment with GNS561 and two positive controls, EAD1 and HCQ, for 16 h. Pearson correlation coefficient between MTOR and LAMP2 was represented using scatter dot plot representation. In (B), (C), (D), (E), (F) and (G), data represent the mean + SEM. For comparison, Student t-test was used for (C), (D), (E), and (G) and one-way ANOVA with Dunnett’s post hoc analysis was performed for (B), (F) and (H). For all studies except (A), n ≥ 3 biological replicates. *represents significant difference, at least p < 0.05.

Article Snippet: Mammalian Cell Lysis Buffer (GE Healthcare, 28–941279), 4-methylumbelliferyl-β-D-6-thiopalmitoylglucoside (Moscerdam, EM06650), Z-VAD-FMK (Bio Techne, FMK001), sorafenib (Santa Cruz Biotechnology, Sc357,801), hexadecanesulfonyl fluoride (HDSF; Santa Cruz Biotechnology, sc-221,708), human PPT1 (OriGene Technologies, TP721098), DC661 (Vagdevi Innoscience), Triton X-100 (Dutscher, 091584B) and cOmpleteTM Protease Inhibitor Cocktail (Roche, 4,693,132,001) were used.

Techniques: Nano Differential Scanning Fluorimetry, Fluorescence, Activity Assay, Control, Western Blot, Staining, Comparison

Journal: Autophagy

Article Title: GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.

doi: 10.1080/15548627.2021.1988357

Figure Lengend Snippet: Figure 7. Schematic representation of molecular and cellular mechanisms involved in the antitumoral activity of GNS561. (A) Schematic illustration showing the stages of untreated tumor progression where autophagy activation and overexpression of PPT1 have been singled out in cell survival and tumor growth. (B) GNS561 compound localizes in lysosomes where it binds and inhibits PPT1 resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, autophagic flux inhibition, alters location of MTOR and leads to lysosomal membrane permeabilization. Finally, all these events induce caspase activation and tumor cell apoptosis.

Article Snippet: Mammalian Cell Lysis Buffer (GE Healthcare, 28–941279), 4-methylumbelliferyl-β-D-6-thiopalmitoylglucoside (Moscerdam, EM06650), Z-VAD-FMK (Bio Techne, FMK001), sorafenib (Santa Cruz Biotechnology, Sc357,801), hexadecanesulfonyl fluoride (HDSF; Santa Cruz Biotechnology, sc-221,708), human PPT1 (OriGene Technologies, TP721098), DC661 (Vagdevi Innoscience), Triton X-100 (Dutscher, 091584B) and cOmpleteTM Protease Inhibitor Cocktail (Roche, 4,693,132,001) were used.

Techniques: Activity Assay, Activation Assay, Over Expression, Inhibition, Membrane

Journal: Nature communications

Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

doi: 10.1038/s41467-023-43650-z

Figure Lengend Snippet: Fig. 1 | PPT1 deficiency protects SLE mice from autoantibodies and nephritis. C57BL/6 J (B6), Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa mice were sacrificed at 16 weeks (n = 5 mice for B6; n = 15 mice for Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa for the entire figure). A Spleen image (left), weight (center) and cell numbers (right) are shown. B Serum anti-DNA antibodies were measured by ELISAs. C Serum anti- RNP/Sm antibodies were measured by ELISAs at 16 weeks. D Serum total IgG were measured by ELISAs. E Glomerulus size was calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 in B6; n = 44 in Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa). F Representative images from kidney immunofluorescence sections are shown (left). IgG (top) and C3 (bottom) staining

Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

Techniques: Staining

Journal: Nature communications

Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

doi: 10.1038/s41467-023-43650-z

Figure Lengend Snippet: Fig. 2 | PPT1 inhibitor HDSF suppresses IFNα in SLE patients and SLE patho- genesis in mice. A PBMCs of SLE patients were treated with DMSO or HDSF overnight. After CpG A stimulation, IFNα levels was evaluated by ELISAs (n = 17 individuals for DMSO/HDSF). B 8-weeks-old B6.Sle1yaa mice (n = 7 mice per group for the rest of figure) were treated with DMSO or HDSF for 8 weeks before sacrifice at 16 weeks. Spleen image (left), weight (center) and cell numbers (right) are shown. C Serum anti-DNA antibodies were measured by ELISAs at indicated weeks of treatment. D Serum anti-RNP/Sm antibodies were measured by ELISAs at the end of treatment. E Serum total IgG were measured by ELISAs at indicated weeks of treatment. F Glomerulus size at 16 weeks is calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 glomeruli per group). G Representative images from kidney immunofluorescence sections are

Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

Techniques: Staining

Journal: Nature communications

Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

doi: 10.1038/s41467-023-43650-z

Figure Lengend Snippet: Fig. 6 | PPT1 depalmitoylates TLR9 in lysosomes. A ABE assays were performed on RAW264.7 cells transduced with mTLR9-HA after 4 h treatment of indicated TLR agonizts. A representative blot (left) and relative mTLR9 S-palmitoylation (right, quantified as the ratio of the mTLR9 output to the calnexin output, n = 3 replicates). B ABE assays were performed on RAW264.7 cells transduced with mTLR7-HA after 4 h treatment of R848. A representative blot (left) and relative mTLR7 S-palmitoylation (right, quantified as the ratio of the mTLR7 output to the calnexin output, n = 3 replicates). C ABE assays were performed on 293 T cells transfected with indicated depalmitoylating enzymes and mTLR9-FLAG (n = 3 replicates).

Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

Techniques: Transduction, Transfection

Journal: Nature communications

Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

doi: 10.1038/s41467-023-43650-z

Figure Lengend Snippet: Fig. 7 | The palmitoylation cycle regulates TLR9 ligand binding. A TNF pro- duction was measured by intracellular staining in CpG B-treated Tlr9-/- RAW264.7 cells transduced with TLR9 Mut2 (n = 4 replicates). B Immunoprecipitation assay with CpG B-biotin were performed on Tlr9-/- RAW264.7 cells transduced with TLR9 WT or Mut2. A representative blot (left) and relative ligand binding (right, calcu- lated as the ratio of CpG B-bound on cleaved mTLR9 to the total mTLR9 in input) are shown (n = 3 replicates). C TNF production was measured by ELISAs in CpG B-treated Zdhhc3+/+ or Zdhhc3-/- RAW264.7 cells transduced with mTLR9-HA (n = 3 replicates). D Immunoprecipitation assays with CpG B-biotin were performed on Zdhhc3+/+ or Zdhhc3-/- RAW264.7 cells transduced with mTLR9-HA (n = 3 replicates). E TNF production was measured by ELISAs in CpG B-treated Ppt1-/- or Ppt1-/-

Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

Techniques: Ligand Binding Assay, Staining, Transduction, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

doi: 10.1016/j.jbc.2024.105641

Figure Lengend Snippet: Reagent resources and list of primers

Article Snippet: Recombinant Human PPT1 , Creative Biomart , Cat#PPT1-367H.

Techniques: Electron Microscopy, Recombinant, Protease Inhibitor, Staining, Isolation, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis

Journal: PLoS ONE

Article Title: Reversible Cysteine Acylation Regulates the Activity of Human Palmitoyl-Protein Thioesterase 1 (PPT1)

doi: 10.1371/journal.pone.0146466

Figure Lengend Snippet: A) PPT1 was co-transfected with HA tagged DHHC enzymes to HEK293 cells. The cells were metabolically labeled with 17-ODYA and PPT1 was immunoprecipitated with anti-PPT1 antibodies. A fluorescent tag was introduced by click chemistry and samples were separated by SDS-PAGE. Fluorescent scanning of the gels detected that PPT1 is palmitoylated by DHHD3 and DHHC7 (top panel, boxed). The relative amount of immunoprecipitated PPT1 is shown (middle panel). The expression of the DHHC enzymes in the cell lysates was verified by immunoblotting with anti-HA antibodies (low panel). B) PPT1 was co-transfected with HA-DHHC3 followed by metabolic labeling, immunoprecipitation of PPT1 and click chemistry. One reaction was treated with hydroxylamine and one reaction with PBS. The addition of hydroxylamine eliminated the fluorescent signal of PPT1 (upper panel) demonstrating that the signal is specific for palmitoylation. Similar amounts of PPT1 were immunoprecipitated and expressed (middle panels). The level of HA-DHHC3 expression in the cell lysates was detected by anti-HA antibodies (lower panel). C) PPT1 is palmitoylated on cysteine residue 6 and can be detected in the cell media. Empty vector, PPT1 or PPT1 C6S were co-expressed with HA-DHHC3 in HEK293, followed by metabolic labeling with 17-ODYA, immunoprecipitation and click chemistry. The control treatment results in a low background fluorescent signal probably due to low levels of expression of endogenous PPT1. The expression and amount of immunoprecipitated PPT1 and PPT1 C6S are similar as detected by immunoblotting with anti-PPT1 antibodies (middle panels). The expression of HA-DHHC3 in was detected by immunoblotting with anti-HA antibodies (lower panel). D) The fluorescence signal intensity for PPT1 and PPT1 C6S were quantified and normalized relative to the amount of immunoprecipitated PPT1 using four independent repeats. The fluorescent signal of PPT1 signal is almost five fold higher than that observed with PPT1 C6S. E) Palmitoylated PPT1 can be detected in cells and media and the signal is reduced following hydroxylamine treatment. PPT1 was co-expressed with HA-DHHC3 in HEK293 cells followed by metabolic labeling both cells and media were collected and PPT1 was immunoprecipitated and labeled using click chemistry. The fluorescent signal of PPT1 palmitoylation is shown in the upper panel. This signal is eliminated by the addition of hydroxylamine. The amount of immunoprecipitated PPT1 and the level of expression in the lysates are shown in the middle panels and in both cell and media by immunoblotting with anti-PPT1 antibodies. The expression of HA-DHHC3 was detected by immunoblotting with anti-HA antibodies in the lower panel. E) Palmitoylation of endogenous PPT1 in human embryonic stem cells. WIBR3 wildtype and 6C, PPT1 knockout clone generated using CRISPR/Cas9 were metabolically labeled with 17-ODYA, followed by immunoprecipitation of PPT1 and click chemistry. We could distinguish fluorescent band appeared in the–HA treatment, that band could be eliminated by hydroxylamine (+HA). The band did not appear in negative control of immunoprecipitation with beads only (-mAB) or in the immunoprecipitation from the PPT1 knockout 6C clone. The lower panel confirms that PPT1 was immunoprecipitated using Western blot.

Article Snippet: Endogenous human PPT1 from hES cells were immunoprecipitated and blotted using mouse anti-human PPT1 (Origene, TA800501).

Techniques: Transfection, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Expressing, Western Blot, Residue, Plasmid Preparation, Control, Fluorescence, Knock-Out, Generated, CRISPR, Negative Control

Journal: PLoS ONE

Article Title: Reversible Cysteine Acylation Regulates the Activity of Human Palmitoyl-Protein Thioesterase 1 (PPT1)

doi: 10.1371/journal.pone.0146466

Figure Lengend Snippet: The localization of PPT1 and PPT1 C6S was examined by co-transfection with fluorescently tagged cellular markers. A-F) Colocalization of PPT1 (A-C) and PPT1 C6S (D-F) to the lysosome marker Lamp1. Both PPT1 and PPT1 C6S are partially localized to the lysosome compartment in COS7 cells. G-L) PPT1 and PPT1 C6S localized also to the Golgi as demonstrated by Golgi marker coexpresssion. M-R) Colocalization of PPT1 and PPT1 C6S to the endoplasmic reticulum is demonstrated using a fluorescent tag attached to the KDEL ER retention signal. The experiments were repeated three times and in each experiment at least 25 cells were imaged.

Article Snippet: Endogenous human PPT1 from hES cells were immunoprecipitated and blotted using mouse anti-human PPT1 (Origene, TA800501).

Techniques: Cotransfection, Marker

Journal: PLoS ONE

Article Title: Reversible Cysteine Acylation Regulates the Activity of Human Palmitoyl-Protein Thioesterase 1 (PPT1)

doi: 10.1371/journal.pone.0146466

Figure Lengend Snippet: The activity of PPT1 in COS7 cells was monitored following the overexpression of either PPT1 or PPT1 C6S, or with the addition of either HA-DHHC3 or HA-DHHC3 dominant negative mutant. The activity of PPT1 was defined as 100%, and the activity in other treatments were calculated accordingly (N = 3). A) In the cell lysates, the addition of HA-DHHC3 reduced PPT1 activity to 81%, while the addition of the dominant negative HA-DHHC3 resulted in 92% activity. The expression of the PPT1 C6S mutant resulted in 142% activity, whereas the co-expression with HA-DHHC3 resulted in activity levels similar to that of the wildtype enzyme (103%). The addition of the HA-DHHC3 dominant negative enzyme elevated the activity to 118%. B) Western blots showing the relative expression of PPT1, HA-DHHC3, and alpha-tubulin (loading control). C) In the cell media the activity of either the single transfected PPT1 or PPT1 C6S differed significantly. However, this effect was eliminated following the co-expression with either active DHHC3 or with its dominant negative mutant. D) The expression of PPT1 in the media was examined by western blot analysis. In the presence of DHHC3 wildtype or dominant negative mutant there was a decrease in the amount of PPT1 in the media. **, p<0.01; ***, p<0.001.

Article Snippet: Endogenous human PPT1 from hES cells were immunoprecipitated and blotted using mouse anti-human PPT1 (Origene, TA800501).

Techniques: Activity Assay, Over Expression, Dominant Negative Mutation, Expressing, Mutagenesis, Western Blot, Control, Transfection

Journal: PLoS ONE

Article Title: Reversible Cysteine Acylation Regulates the Activity of Human Palmitoyl-Protein Thioesterase 1 (PPT1)

doi: 10.1371/journal.pone.0146466

Figure Lengend Snippet: The Km and Vmax of PPT1 and PPT1 C6S were calculated using the Michaelis-Menten equation following plotting the results of enzymatic activity obtained with different concentrations of the substrate (N = 9) A) Michaelis-Menten graph derived from the cell lysates. B) Michaelis-Menten graph derived from the cell media.

Article Snippet: Endogenous human PPT1 from hES cells were immunoprecipitated and blotted using mouse anti-human PPT1 (Origene, TA800501).

Techniques: Activity Assay, Derivative Assay

Journal: Neurobiology of disease

Article Title: Defective anterograde protein-trafficking contributes to endoplasmic reticulum-stress in a CLN1 disease model.

doi: 10.1016/j.nbd.2025.106890

Figure Lengend Snippet: Fig. 3. Ppt1-deficiency impairs interaction of Sar1 with inner layers of COPII vesicles. (A)Western blot analysis of Sec12 in the total homogenates of cortical tissues from 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4 animals in each group, p ≤0.05; NS = non-significant). (B) Western blot analysis of Sec12 in the ER fraction isolated from the cortical tissues of 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4). (C) Pull down experiments using anti-Sar1, which pulled down Sec12 from total homogenates of cortical tissues from 6-month-old WT and Cln1−/−mice (n = 4). (D) Anti-Sec12 pulls down Sar1 from total homogenates of cortical tissues from 6-month-old WT and Cln1 −/−mice (n = 4); (E) Colocalization of Sec12 with Sar1 in control and CLN1 patient fibroblasts; Colocalization between Sec12 and Sar1 was assessed using the Manders colocalization coefficients (n = 24 cells); (F) Anti-Sec23A pulls down Sar1 in total homogenates of cortical lysates from 6 month old WT and Cln1−/−mice (n = 4); (G) Anti-Sar1 pulls down Sec23A in total cortical lysates from 6 month old WT and Cln1−/−mice (n = 4); (H) Antibody to Sec24A pulls down Sar1 from total cortical lysates from 6 month old WT and Cln1 −/−mice (n = 4); (I) Antibody to Sar1 pulls down Sec24A from total cortical lysates from 6 month old WT and Cln1 −/−mice (n = 4). The data are presented as the mean ± SD and the ‘n’ numbers denote the number of biological replicates for each experiment. *p-values ≤0.05 were considered statistically significant.

Article Snippet: Myc-DDK tagged mouse Sar1 (OriGene Technologies Inc., Rockville, Cat#MR201967) and Mouse Flag-tagged CLN8 (Genecopoeia, Rockville, Cat# ExMm06485-M39) and PPT1 (Myc-DDK-tagged)-Human palmitoylprotein thioesterase-1 (OriGene Technologies Inc., Rockville, Cat# SKU RC203278) plasmids were used for the transfection studies.

Techniques: Western Blot, Isolation, Control

Journal: Neurobiology of disease

Article Title: Defective anterograde protein-trafficking contributes to endoplasmic reticulum-stress in a CLN1 disease model.

doi: 10.1016/j.nbd.2025.106890

Figure Lengend Snippet: Fig. 5. Anterograde trafficking of Cln8-protein is dysregulated in Cln1−/−mice. (A) Western blot analysis of Cln8-protein in the total homogenates of cortical tissues from 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4 animals in each group, p ≤0.05; NS = non-significant.) (B) Colocalization of CLN8 with ER marker, Calnexin, in control, CLN1 patient fibroblasts and CLN1 patient fibroblast transfected with CLN1/PPT1-cDNA construct was evaluated using Manders’ colocalization coefficients (n = 24). (C) Western blot analysis of Cln2-protein in total homogenates of cortical tissues from 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4 animals in each group, p ≤0.05; NS = non-significant). (D) Western blot analysis of Cln2- protein in the ER fractions from the cortical tissues of 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4, p ≤0.05). (E) Colocalization of ER marker, calnexin, with CLN2 in control and CLN1 patient fibroblasts was evaluated using Mander’s colocalization coefficients (n = 24 cells). (F) Proximity ligation assay to confirm the interaction of Cln8 with Cln2 in control and CLN1 patient fibroblasts (n = 24 cells), p ≤0.05; (G) Western blot analysis of Cln8-protein in the lysosomal fractions from the cortical tissues of 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4, p ≤0.05). (H) Western blot analysis of Cln2-protein in the lysosome fraction from the cortical tissues of 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4, p ≤0.05). The data are presented as the mean ± SD, and the ‘n’ numbers denote the number of biological replicates for each experiment. *p-values ≤0.05 were considered statistically significant.

Article Snippet: Myc-DDK tagged mouse Sar1 (OriGene Technologies Inc., Rockville, Cat#MR201967) and Mouse Flag-tagged CLN8 (Genecopoeia, Rockville, Cat# ExMm06485-M39) and PPT1 (Myc-DDK-tagged)-Human palmitoylprotein thioesterase-1 (OriGene Technologies Inc., Rockville, Cat# SKU RC203278) plasmids were used for the transfection studies.

Techniques: Western Blot, Marker, Control, Transfection, Construct, Proximity Ligation Assay

Journal: Neurobiology of disease

Article Title: Defective anterograde protein-trafficking contributes to endoplasmic reticulum-stress in a CLN1 disease model.

doi: 10.1016/j.nbd.2025.106890

Figure Lengend Snippet: Fig. 6. The unfolded protein response leads to ER stress in WT and Cln1−/−mice. (A) Level of unfolded protein response elements, GRP78, XBP1 and ATF6 in the cortical homogenate of 6-month-old WT and Cln1−/−mice. (B) Analysis of ER stress Transcription factor activation profiling in 6-month-old WT and Cln1−/−mice. (C) Enzymatic activity of Ppt1 in Control HEK 293 T, CLN1−/−HEK 293 T cells and CLN1−/−HEK 293 T cells+ PPT1 cDNA construct. (n = 4, p ≤0.05). (D) Level of GRP-78, (E) XBP-1 and (F) ATF-6 in in Control HEK 293 T, CLN1−/−HEK 293 T cells and CLN1−/−HEK 293 T cells+ PPT1 cDNA construct. (n = 4, p ≤0.05). (1- Control HEK293T cells, 2-CLN1−/− HEK293T cells and 3-CLN1−/− HEK293T cells + myc tagged- PPT1 construct). The data are presented as the mean ± SD and the ‘n’ numbers denote the number of biological replicates for each experiment. *p-values ≤0.05 were considered statistically significant and NS is non -significant.

Article Snippet: Myc-DDK tagged mouse Sar1 (OriGene Technologies Inc., Rockville, Cat#MR201967) and Mouse Flag-tagged CLN8 (Genecopoeia, Rockville, Cat# ExMm06485-M39) and PPT1 (Myc-DDK-tagged)-Human palmitoylprotein thioesterase-1 (OriGene Technologies Inc., Rockville, Cat# SKU RC203278) plasmids were used for the transfection studies.

Techniques: Activation Assay, Activity Assay, Control, Construct

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 4. PPT1 expression is upregulated in senescent macrophages. A) Volcano plot of differentially expressed proteins of Young and Aged groups in rats heart. B) KEGG enrichment top 20. C) Heat map of differentially expressed proteins in the lysosomal pathway. D,E) Representative images and statistical analysis of IHC staining with PPT1 antibody. Magnification: 400×, scale bar = 100 μm. F) Statistical analysis of PPT1 mRNA level. G) Representative images and statistical analysis of PPT1 protein level. H) UMAP plots of heart macrophage from 27 healthy donors. I) A heatmap showing the markers ≈7 types of macrophages. J) Stacked graph of cell proportions. K) Violin plots of inflammatory response scores in different subgroups. L) Violin plots of the M1 scores of different macrophage subsets. M) Violin plots of PPT1 expression in different monocyte-macrophage subsets. N) Inflammatory response score. O) M1 score. P) Representative figure of the immunofluorescence of CD68, PPT1, and DAPI in heart. Magnification: 200×, scale bar = 20 μm. Q,R) Representative immunofluorescence images and statistical analysis of PPT1 intensity. Magnification: 200×, scale bar = 20 μm. S) Statistical analysis of PPT1 mRNA level in BMDM. T) Representative graphs and statistical analysis of PPT1 at protein level in BMDM. (n = 3–6, data are expressed as mean ± SEM, *p < 0.05, **p < 0.01 vs the Young group).

Article Snippet: The PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

Techniques: Expressing, Immunohistochemistry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 5. Transgenic knockout of macrophage PPT1 improves cardiac inflammatory infiltration and myocardial fibrosis in D-gal induce-aged mice. A) Schematic of the experimental design. B) Representative echocardiographic graphs. C–J) Echocardiographic measurements of LVEF, LVFS, LVIDd, LVIDs, LVPWd, LVPWs, IVSd, and IVSs. K) H&E staining of left ventricles. Magnification: 200×, scale bar = 50 μm. L) Masson’s staining of left ventricles. Magnification: 200×, scale bar = 50 μm. M,N) Representative images of IHC staining with MCP-1 and 𝛼-SMA antibody. Magnification: 200×, scale bar = 50 μm. O) Statistical graphs of Masson’s staining. P,Q) Statistical graphs of IHC staining with MCP-1 and 𝛼-SMA antibody. R) Representative images of the immunofluorescence of CD68, PPT1, and DAPI in mice heart. Magnification: 200×, scale bar = 20 μm. S) Statistical analysis of COL3A1, COL1A1, PPT1, IL-6, and TNF-𝛼mRNA level. T,U) Representative images and statistical analysis of COL3A1, COL1A1, PPT1, IL-6, and TNF-𝛼at protein level. (n = 5–6, data are expressed as mean ± SEM, **p < 0.01 vs the WT group; #p < 0.05, ##p < 0.01 vs the D-gal group).

Article Snippet: The PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

Techniques: Transgenic Assay, Knock-Out, Staining, Immunohistochemistry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 6. IPA inhibits PPT1 expression and reduces the secretion of inflammatory factors in aging macrophages. A,B) Representative images and statistical graphs of the immunofluorescence of iNOS. Magnification: 200×, scale bar = 20 μm. C) Statistical analysis of IL-6 and TNF-𝛼at mRNA level. D,E) Representative images and statistical analysis of IL-6 and TNF-𝛼at protein level. F,G) Representative images and statistical graphs of the immunofluorescence of iNOS. Magnification: 200×, scale bar = 20 μm. H) Statistical analysis of IL-6 and TNF-𝛼at mRNA level. I,J) Representative images and statistical analysis of IL-6 and TNF-𝛼at protein level. (n = 3–6, data are expressed as mean ± SEM, **p < 0.01 vs the Young or Vector group; #p < 0.05, ##p < 0.01 vs the Aged or oe-PPT1 group).

Article Snippet: The PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

Techniques: Expressing, Plasmid Preparation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 7. IPA inhibits PPT1 in aged macrophages and alleviates collagen deposition in fibroblasts. A) Schematic of the experimental design. B,C) Representative figures and statistical graphs of the immunofluorescence of 𝛼-SMA. Magnification: 200×, scale bar = 20 μm. D) Statistical analysis of COL3A1, COL1A1 and 𝛼-SMA at mRNA level. E,F) Representative images and statistical analysis of COL3A1, COL1A1 and 𝛼-SMA at protein level. G,H) Representative Figure and statistical graphs of the immunofluorescence of 𝛼-SMA. Magnification: 200×, scale bar = 20 μm. I) Statistical analysis of COL3A1, COL1A1 and 𝛼-SMA at mRNA level. J,K) Representative figures and statistical analysis of COL3A1, COL1A1 and 𝛼-SMA at protein level. (n = 3– 6, data are expressed as mean ± SEM, *p < 0.5, **p < 0.01 vs the Young–CM or Vector–CM group; #p < 0.5, ##p < 0.01 vs the Aged–CM or oe-PPT1–CM group).

Article Snippet: The PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

Techniques: Plasmid Preparation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 8. IPA inhibits PPT1 in elderly macrophages in vitro and reduces myocardial fibrosis in vivo. A) Schematic of the experimental design. B,C) Rep- resentative graphs and statistical graph of flow cytometry. D) Representative echocardiographic graphs. E–L) Echocardiographic measurements of LVEF, LVFS, LVIDd, LVIDs, LVPWd, LVPWs, IVSd, and IVSs. M) H&E staining of left ventricles. Magnification: 200×, scale bar = 50 μm. N) Masson’s staining of left ventricles. Magnification: 200×, scale bar = 50 μm. O,P) Representative images of IHC staining with MCP-1 and 𝛼-SMA antibody. Magnification: 200×, scale bar = 50 μm. Q) Statistical graphs of Masson’s staining. R,S) Statistical graphs of IHC staining with MCP-1 and 𝛼-SMA antibody. T) Statistical analysis of COL3A1, COL1A1, IL-6, and TNF-𝛼at mRNA level. U,V) Representative images and statistical analysis of COL3A1, COL1A1, IL-6, and TNF-𝛼 at protein level. (n = 3–6, data are expressed as mean ± SEM, *p < 0.05, **p < 0.01 vs the Young group; #p < 0.05, ##p < 0.01 vs the Aged group; &p < 0.05, &&p < 0.01 vs the Aged+sh-PPT1 group).

Article Snippet: The PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

Techniques: In Vitro, In Vivo, Cytometry, Staining, Immunohistochemistry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 10. cGAS-STING pathway is involved in IPA-induced inhibition of PPT1. A) Statistical graph of cGAS and STING mRNA levels in macrophages. B,C) Representative images and statistical graph of cGAS and STING at protein levels in macrophages. D,E) Representative images and statistical graph of cGAS and STING at protein levels in vitro. F) Schematic diagram of IPA. G–J) Representative images and statistical graph of PPT1 immunofluorescence intensity in M1 macrophages after treated with RU.521 or C176. Magnification: 200×, scale bar = 20 μm. K,L) Representative images and statistical graph of PPT1 protein levels in M1 macrophages after treated with RU.521 or C176. M) Graphical abstract. (n = 3–6, data are expressed as mean ± SEM, *p < 0.05, **p < 0.01 vs the Young or Control group; #p < 0.05, ##p < 0.01 vs the Aged or LPS/IFN-𝛾group).

Article Snippet: The PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

Techniques: Inhibition, In Vitro, Control

Journal: Autophagy

Article Title: Divergent effects of acute and chronic PPT1 inhibition in melanoma

doi: 10.1080/15548627.2024.2403152

Figure Lengend Snippet: Cytotoxicity observed by acute but not chronic PPT1 inhibition. (A) Immunoblot demonstrating PPT1 knockout in A375P sg PPT1 Hug1 and Hug3 clones compared to A375P sg NT cells. sg: small guide RNA; Hug: human guide; NT: non-target. (B) Van Diggelen PPT1 biochemical assay in the indicated cells. n = 3, repeated twice. **** P ≤ 0.0001; one way ANOVA test. (C) Cell growth at 4 days after plating the indicated cell lines at 1X10 5 . n = 3, repeated 3 times, ns: nonsignificant, one way ANOVA. (D) Immunoblot demonstrating PPT1 knockdown in A375P cells after doxycycline (Dox) induction of Cas9 expression. Cells with (+) or without (-) Dox treatment were collected on the indicated days after the start of treatment. (E) A375P iCas9 cell growth at 6 days after Dox treatment. (F) A375P iCas9 Van Diggelen PPT1 biochemical assay at 6 days after Dox treatment. (G) Immunoblot of A375P sg NT cells transfected with siNT or si PPT1 for 48 h. (H) Van Diggelen PPT1 biochemical assay in indicated cells 48 h after siRNA transfection. n = 3. (I) Cell growth 48 h after siRNA transfection, n = 3. (J) Van Diggelen PPT1 biochemical assay in A375P cells treated with 3 µm DC661 for 24 h, n = 3. (K) Cell number after treatment with 3 µm DC661 for 24 h. All cell growth assays and enzyme assays were performed in triplicate. * P ≤ 0.05; **** P ≤ 0.0001; one way ANOVA was used when more than 2 groups were compared (B, C, and F), two-tailed unpaired t test was used when 2 groups were compared (E, H-K).

Article Snippet: Primary antibodies included PPT1 for human samples (OriGene, CF800490; 1:1000), PPT1 for mouse samples (GeneTex, GTX110677; 1:5000), Cas9 (Cell Signaling Technology, 65832S; 1:1000) and ACTB/β-actin (Cell Signaling Technology, 3700S; 1:5000).

Techniques: Inhibition, Western Blot, Knock-Out, Clone Assay, Knockdown, Expressing, Transfection, Two Tailed Test

Journal: Autophagy

Article Title: Divergent effects of acute and chronic PPT1 inhibition in melanoma

doi: 10.1080/15548627.2024.2403152

Figure Lengend Snippet: Proteomic changes during acute versus chronic PPT1 inhibition. (A) MS abundance of PPT1 protein in A375P cells in which PPT1 is depleted by either CRISPR-Cas9, or siRNA. NT: non-target, sg: small guide RNA. (B) Unsupervised hierarchical cluster comparing proteomes of constitutive Cas9 (sg) and acute siRNA (si) PPT1 inhibition (C) Volcano plots of sg PPT1 vs. sg NT and si PPT1 vs. si NT , (D) Volcano plot of DC661 vs. control (DMSO), reanalyzed from reference 12. Red = q-value ≤0.05 and ≥ 1.5 fold increase; Blue = q-value ≤0.05 and ≥ 1.5 fold decrease. (E) Venn diagram demonstrating overlapping proteins that are significantly increased with sg PPT1 vs. sg NT , si PPT1 vs. si NT , and DC661 vs. control (DMSO).

Article Snippet: Primary antibodies included PPT1 for human samples (OriGene, CF800490; 1:1000), PPT1 for mouse samples (GeneTex, GTX110677; 1:5000), Cas9 (Cell Signaling Technology, 65832S; 1:1000) and ACTB/β-actin (Cell Signaling Technology, 3700S; 1:5000).

Techniques: Inhibition, CRISPR, Control

Journal: Autophagy

Article Title: Divergent effects of acute and chronic PPT1 inhibition in melanoma

doi: 10.1080/15548627.2024.2403152

Figure Lengend Snippet: Top 20 proteins significantly increased and decreased in chronic and acute genetic PPT1 inhibition (q ≤ 0.05).

Article Snippet: Primary antibodies included PPT1 for human samples (OriGene, CF800490; 1:1000), PPT1 for mouse samples (GeneTex, GTX110677; 1:5000), Cas9 (Cell Signaling Technology, 65832S; 1:1000) and ACTB/β-actin (Cell Signaling Technology, 3700S; 1:5000).

Techniques: Inhibition

Journal: Autophagy

Article Title: Divergent effects of acute and chronic PPT1 inhibition in melanoma

doi: 10.1080/15548627.2024.2403152

Figure Lengend Snippet: Top 20 proteins significantly increased and decreased in si PPT1 /si NT vs sg PPT1 /sg NT (q ≤ 0.05).

Article Snippet: Primary antibodies included PPT1 for human samples (OriGene, CF800490; 1:1000), PPT1 for mouse samples (GeneTex, GTX110677; 1:5000), Cas9 (Cell Signaling Technology, 65832S; 1:1000) and ACTB/β-actin (Cell Signaling Technology, 3700S; 1:5000).

Techniques:

Journal: Autophagy

Article Title: Divergent effects of acute and chronic PPT1 inhibition in melanoma

doi: 10.1080/15548627.2024.2403152

Figure Lengend Snippet: Pathway alterations in acute and chronic PPT1 inhibition. (A) Top enriched canonical pathways identified by DAVID pathway analysis when comparing significantly increasedor decreased proteins in sg PPT1 vs. sg NT . (B) Top enriched pathways identified when comparing significantly increased or decreased proteins in si PPT1 vs. si NT . (C) Top enriched pathways identified when comparing normalized si PPT1 vs. sg PPT1 . Enriched pathways with Fisher’s exact test p-value ≤0.01 were considered significant. (D) Bodipy-C11 analysis indicated blunted ferroptosis induction in A375P sg NT and sg PPT1 after treatment with 0.5 μM RSL3. One way ANOVA was used to compare the groups. * P ≤ 0.05; *** P ≤ 0.001 (E) Bodipy-C11 analysis after 0.5 μM RSL3 treatment indicated enhanced ferroptosis induction in si PPT1 treated A375P compared to cells that received si NT . One way ANOVA was used to compare the groups. *** P ≤ 0.0001; **** P ≤ 0.0001. RFU: relative fluorescence units.

Article Snippet: Primary antibodies included PPT1 for human samples (OriGene, CF800490; 1:1000), PPT1 for mouse samples (GeneTex, GTX110677; 1:5000), Cas9 (Cell Signaling Technology, 65832S; 1:1000) and ACTB/β-actin (Cell Signaling Technology, 3700S; 1:5000).

Techniques: Inhibition, Fluorescence

Journal: Autophagy

Article Title: Divergent effects of acute and chronic PPT1 inhibition in melanoma

doi: 10.1080/15548627.2024.2403152

Figure Lengend Snippet: A Ppt1 conditional knockout mouse. (A and B)Schema for generating a Ppt1 conditional knockout allele using 2 CRISPR-Cas9 strategies that were pursued concurrently. (A) Sequential LoxP site insertions Cas9 mRNA, 2 guide RNAs (sgRNA) and 2 repair templates (LoxP sites represented by arrowheads – red 3’ LoxP, yellow 5’ LoxP) were injected into the pronucleus of fertilized C57Bl/6J oocytes. Mice with the 3’ LoxP site were obtained and then bred together. Sperm from a mouse homozygous for the 3’LoxP site was used to in vitro fertilize oocytes which were then injected with Cas9 mRNA, the 5’ guide RNA, and the 5’ LoxP repair template. Black arrowheads indicate primer locations on either side of the LoxP insertion sites used for screening in (C). (B) Easi-CRISPR – Cas9 mRNA, 2 guide RNAs, and a long single stranded repair template with both LoxP sites and exon 1 were injected into fertilized oocytes. Black arrowheads indicate primer locations on either side of the LoxP insertion sites used for screening in (D). (C) PCR screening of the sequential founders with primer pairs Ppt1 F3/R3 and 3’ Ppt1 F3/R3. Only #6 (red number) was positive for both 5’ and 3’ LoxP sites. (D) PCR screening of Easi-CRISPR founders with primer pairs Ppt1 F7/R7 and Ppt1 205 F/611 R. Founders #25, 32, 34, 37 & 39 (red numbers) appeared to have both 5’ LoxP and 3’ LoxP sites. (E) Representative images of 4-HT induced Braf mutant pten null melanoma tumors with the indicated Ppt1 genotypes. (F) PCR of the entire 1381 bp Ppt1 F3/611 R amplicon (diagram indicates location of the primers) of DNA isolated from tails and from 4-HT induced melanoma tumors demonstrates deletion of Ppt1 exon 1 by Cre Recombinase in ppt1 fl/fl and Ppt1 f l/WT tumors in both line 6 and line 25, but not in wild-type Ppt1 mice. Expected bands for deletion of Ppt1 exon 1 were 384 bp for line 6 and 847 bp for line 25. G: Genomic DNA, T: tumor DNA.

Article Snippet: Primary antibodies included PPT1 for human samples (OriGene, CF800490; 1:1000), PPT1 for mouse samples (GeneTex, GTX110677; 1:5000), Cas9 (Cell Signaling Technology, 65832S; 1:1000) and ACTB/β-actin (Cell Signaling Technology, 3700S; 1:5000).

Techniques: Knock-Out, CRISPR, Injection, In Vitro, Mutagenesis, Amplification, Isolation

Journal: Autophagy

Article Title: Divergent effects of acute and chronic PPT1 inhibition in melanoma

doi: 10.1080/15548627.2024.2403152

Figure Lengend Snippet: Ppt1 is dispensable for melanoma tumor growth. (A) Time to tumor formation by genotype in line 6 and line 25 (WT n = 17; Ppt1 fl/WT n = 38; ppt1 fl/fl n = 49) graphed is time to first appearance of black dots after 4-HT treatment, and time to coalescence (merging of the tumor dots). Representative images are shown. (B) Tumor growth measured weekly after 4-HT treatment in both lines by genotype (WT n = 43; Ppt1 fl/WT n = 65; ppt1 fl/fl n = 65); ANOVA comparing tumor volumes on the last day. (C) Kaplan-Meier survival curve (time to 2000 mm 3 tumor volume) by genotype in both lines. (D) Van Diggelen PPT1 enzyme assay in tumor samples from 3 mice of each Ppt1 genotype with duplicate technical replicates. (E) Representative H&E slides of melanoma tumors of each genotype at 100X magnification. (F) Immunoblot demonstrating Ppt1 knockout in YUMM1.7 sg Ppt1 Msg1 and Msg3 clones compared to YUMM1.7 sg NT cells. sg: small guide RNA; Msg: mouse guide; NT: non-target. (G) Van Diggelen PPT1 biochemical assay in the indicated cells. n = 3, repeated twice. (H) Cell growth at 3 days after plating the indicated cell lines at 1X10 5 . n = 3. (I) Bodipy C-11 fluorescence of YUMM1.7 cells treated as indicated for 24 h demonstrated a blunted response to RSL3 ferroptosis induction in sg Ppt1 cells. One way ANOVA test was used to compare multiple groups. ns: nonsignificant,* p ≤ 0.05, ** p ≤ 0.005, *** P ≤ 0.001; **** P ≤ 0.0001; RFU: relative fluorescence units.

Article Snippet: Primary antibodies included PPT1 for human samples (OriGene, CF800490; 1:1000), PPT1 for mouse samples (GeneTex, GTX110677; 1:5000), Cas9 (Cell Signaling Technology, 65832S; 1:1000) and ACTB/β-actin (Cell Signaling Technology, 3700S; 1:5000).

Techniques: Enzymatic Assay, Western Blot, Knock-Out, Clone Assay, Fluorescence

Journal: Autophagy

Article Title: Divergent effects of acute and chronic PPT1 inhibition in melanoma

doi: 10.1080/15548627.2024.2403152

Figure Lengend Snippet:

Article Snippet: Primary antibodies included PPT1 for human samples (OriGene, CF800490; 1:1000), PPT1 for mouse samples (GeneTex, GTX110677; 1:5000), Cas9 (Cell Signaling Technology, 65832S; 1:1000) and ACTB/β-actin (Cell Signaling Technology, 3700S; 1:5000).

Techniques: Sequencing

Journal: Autophagy

Article Title: Divergent effects of acute and chronic PPT1 inhibition in melanoma

doi: 10.1080/15548627.2024.2403152

Figure Lengend Snippet:

Article Snippet: Primary antibodies included PPT1 for human samples (OriGene, CF800490; 1:1000), PPT1 for mouse samples (GeneTex, GTX110677; 1:5000), Cas9 (Cell Signaling Technology, 65832S; 1:1000) and ACTB/β-actin (Cell Signaling Technology, 3700S; 1:5000).

Techniques: Sequencing