Journal: Data in Brief

Article Title: Quantitative analysis of PPT1 interactome in human neuroblastoma cells

doi: 10.1016/j.dib.2015.05.016

Figure Lengend Snippet: Global network analysis of PPT1 IP. Functional coupling of PPT1 IP was analysed by FunCoup database v.3.0 ( http://FunCoup.sbc.su.se/ ). Based on 24 input genes (including PPT1 and PPT1 IP), 65 functionally coupled pairs with a very high confidence score (>0.8, scale 0−1) were identified. Dots, dashed and solid lines; indicate scores between (0.82–0.89), (0.9–0.95) and (>0.95), respectively. Details are provided in , Supporting Information.

Article Snippet: Cells were cultured in DMEM medium supplemented with 600 μg/ml G-418 to select for clones stably expressing the constructs. qPCR experiments using inventoried Taqman assay (Applied Biosystems, Life Technologies) were performed to assess the expression of CLN1 mRNA (Hs00165579_m1); GAPDH (Hs99999905_m1) was used as reference endogenous gene. cDNAs were amplified in a ABI 7500Fast real time PCR.

Techniques: Functional Assay

Journal: Data in Brief

Article Title: Quantitative analysis of PPT1 interactome in human neuroblastoma cells

doi: 10.1016/j.dib.2015.05.016

Figure Lengend Snippet: PPT1 IP association terms network. PPT1 interacting partners served as inputs into Ingenuity Pathways analyses ( https://analysis.ingenuity.com/ ). Three terms: inhibition of organismal death ( Z -score −3.109), Movement disorders ( Z -score −2.209) and concentration of lipid ( Z -score −2.133) were predicted to be altered in the PPT1 network.

Article Snippet: Cells were cultured in DMEM medium supplemented with 600 μg/ml G-418 to select for clones stably expressing the constructs. qPCR experiments using inventoried Taqman assay (Applied Biosystems, Life Technologies) were performed to assess the expression of CLN1 mRNA (Hs00165579_m1); GAPDH (Hs99999905_m1) was used as reference endogenous gene. cDNAs were amplified in a ABI 7500Fast real time PCR.

Techniques: Inhibition, Concentration Assay

Journal: Data in Brief

Article Title: Quantitative analysis of PPT1 interactome in human neuroblastoma cells

doi: 10.1016/j.dib.2015.05.016

Figure Lengend Snippet: PPT1 interaction network. PPT1 interaction partners were linked to PPT1 using known physical and genetic interactions, pathway data and utilising knowledge of predicted interactions. The network was filtered according to GO BP criteria using GeneMANIA algorithm ( www.genemania.org ). Overall, 74 nodes of the network could be connected using 88 physical and 69 genetic interactions, 124 pathway and 133 predicted links. Pathway information from Pathway commons (Consolidated-Pathways-2013) was used to facilitate additional 16 links with varying weights (0.15–16.83%) to the PPT1 network. Nodes enriched with selected GO BP terms are indicated. Detailed information about the parameters of the network is presented in .

Article Snippet: Cells were cultured in DMEM medium supplemented with 600 μg/ml G-418 to select for clones stably expressing the constructs. qPCR experiments using inventoried Taqman assay (Applied Biosystems, Life Technologies) were performed to assess the expression of CLN1 mRNA (Hs00165579_m1); GAPDH (Hs99999905_m1) was used as reference endogenous gene. cDNAs were amplified in a ABI 7500Fast real time PCR.

Techniques:

Journal: Redox Biology

Article Title: Melatonin protects aged oocytes from depalmitoylation-mediated quality reduction by promoting PPT1 degradation and antioxidation

doi: 10.1016/j.redox.2025.103510

Figure Lengend Snippet: The expression of PPT1 in the ovary is correlated with age. (A) Western blot analysis showing PPT1 expression in ovaries from control and aged mice. (B) Western blot analysis showing PPT1 expression in mouse oocytes from control and aged mice. (C) Immunofluorescence staining was used to assess the subcellular localization of PPT1. Green, PPT1; blue, chromatin. (D) Immunofluorescence colocalization analysis of tubulin and PPT1 in mouse oocytes during the MI phase. Red, PPT1; green, tubulin; blue, chromatin. (E) The relative mRNA level of PPT1 in control and PPT1-mRNA-injected (called the oePPT1 group) oocytes was determined by quantitative RT-PCR. The mRNA level in control oocytes was set to 1. (F) Western blot analysis showing PPT1 expression in oocytes from mice in the control, oePPT1 and oePPT1+mel groups. (G) Pb1 extrusion rate in the control (n = 135), oePPT1 (n = 173) and oePPT1+mel (n = 218) groups. (H) Western blot analysis showing PPT1 expression in young (n = 3) and aged (n = 3) donor ovaries. (I) The results of PPT1 ELISA showed that the PPT1 protein expression in follicular fluid of young (n = 21), aged-control (n = 13) and aged-DOR (n = 25) donors. β-Actin served as a loading control for Western blotting. The data are presented as the means ± SDs from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The membranes were blocked with 5 % BSA at room temperature for 1 h and incubated with the following primary antibodies diluted to the appropriate concentration in a shaker at 4 °C overnight: anti-PPT1 antibody (1:3000), anti-actin antibody (Proteintech, 66009-1-Ig; 1:5000) and anti-tubulin antibody (Cell Signaling Technology, 2148s, 1:5000).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining, Injection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Redox Biology

Article Title: Melatonin protects aged oocytes from depalmitoylation-mediated quality reduction by promoting PPT1 degradation and antioxidation

doi: 10.1016/j.redox.2025.103510

Figure Lengend Snippet: Effects of melatonin on PPT1 expression in oocytes and ovaries during maternal aging. (A) Western blot analysis showing PPT1 expression in oocytes in the control, H 2 O 2 and H 2 O 2 + mel groups. (B) Timeline of melatonin administration to mice. (C) The level of melatonin in the blood serum of young mice (n = 9), aged mice (n = 10) and melatonin-treated mice (n = 7). (D) Western blot analysis showing PPT1 expression in oocytes from control, aged and aged + mel/i.g. mice. (E) Western blot analysis showing PPT1 expression in oocytes from control, aged and melatonin-treated mice. (F) Binding mode of melatonin to PPT1 by molecular docking. (G) PPT1, mouse (HEK293, His) can bind melatonin with an affinity constant of 0.007511 M as determined in SPR assay. (H) Oocytes in the control, aged and aged + mel groups were collected and stained to visualize lysosome (red) and chromosomes (blue). Scale bar, 20 μm. (I) The relative fluorescence intensity in the control (n = 21), aged (n = 25) and aged + mel (n = 29) groups based on the area of Lamp1 signals.(J) The expression level of PPT1 in HEK293T cells treated with PPT1-HA, oePPT1 plus melatonin, or additionally treated with CQ or CHX. β-Actin served as a loading control for Western blotting. The data are presented as the means ± SDs from three independent experiments. ∗ p < 0.05, ∗∗∗∗ p < 0.0001.

Article Snippet: The membranes were blocked with 5 % BSA at room temperature for 1 h and incubated with the following primary antibodies diluted to the appropriate concentration in a shaker at 4 °C overnight: anti-PPT1 antibody (1:3000), anti-actin antibody (Proteintech, 66009-1-Ig; 1:5000) and anti-tubulin antibody (Cell Signaling Technology, 2148s, 1:5000).

Techniques: Expressing, Western Blot, Control, Binding Assay, SPR Assay, Staining, Fluorescence

Journal: Redox Biology

Article Title: Melatonin protects aged oocytes from depalmitoylation-mediated quality reduction by promoting PPT1 degradation and antioxidation

doi: 10.1016/j.redox.2025.103510

Figure Lengend Snippet: Global S-palmitoylation levels in oocytes were regulated by PPT1 and melatonin. (A) Schematic of ABE for detecting global protein S-palmitoylation. (B) Analysis of the palmitoylation levels of proteins extracted from oocytes from control, aged and aged + mel/i.g. mice. (C) Analysis of the palmitoylation levels of proteins extracted from control and aged oocytes with or without melatonin treatment. (D) Analysis of the palmitoylation levels of proteins extracted from control, H 2 O 2 and H 2 O 2 + mel oocytes. (E) Analysis of the palmitoylation levels of proteins extracted from oocytes in the control, oePPT1 and oePPT1 + mel groups. (F) Analysis of the palmitoylation levels of proteins extracted from control and 2BP-treated (called the 2BP) oocytes. (G) Representative phase contrast images of MII oocytes from the control and 2BP groups. Scale bar, 50 μm. (H) Pb1 extrusion rates of control (n = 240) and 2BP-treated oocytes (n = 301). Actin served as a loading control for ABE assay. The data are presented as the means ± SDs from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The membranes were blocked with 5 % BSA at room temperature for 1 h and incubated with the following primary antibodies diluted to the appropriate concentration in a shaker at 4 °C overnight: anti-PPT1 antibody (1:3000), anti-actin antibody (Proteintech, 66009-1-Ig; 1:5000) and anti-tubulin antibody (Cell Signaling Technology, 2148s, 1:5000).

Techniques: Control

Journal: Redox Biology

Article Title: Melatonin protects aged oocytes from depalmitoylation-mediated quality reduction by promoting PPT1 degradation and antioxidation

doi: 10.1016/j.redox.2025.103510

Figure Lengend Snippet: 2BP and overexpressed PPT1 affects mouse oocyte spindle organization. (A) Oocytes in the control and 2BP groups were collected and stained to visualize spindles (green) and chromosomes (blue). Scale bar, 20 μm. (B) Quantitative analysis of control (n = 119) and 2BP (n = 101) oocytes with spindle defects or chromosome misalignment. (C) Quantitative analysis of spindle width in control (n = 21) and 2BP (n = 24) oocytes. (D) Oocytes from the control, oePPT1 and oePPT1 + mel groups were collected and stained to visualize spindles (green) and chromosomes (red). Aberrant spindles (arrows) and misaligned chromosomes (arrowheads) were readily observed in oePPT1 oocytes. Scale bar, 20 μm. (E) Quantitative analysis of oocytes from the control (n = 116), oePPT1 (n = 104) and oePPT1 + mel (n = 120) groups with spindle defects or chromosome misalignment. (F) Quantitative analysis of spindle width in control (n = 26), oePPT1 (n = 28) and oePPT1 + mel (n = 21) oocytes. The data are presented as the means ± SDs from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The membranes were blocked with 5 % BSA at room temperature for 1 h and incubated with the following primary antibodies diluted to the appropriate concentration in a shaker at 4 °C overnight: anti-PPT1 antibody (1:3000), anti-actin antibody (Proteintech, 66009-1-Ig; 1:5000) and anti-tubulin antibody (Cell Signaling Technology, 2148s, 1:5000).

Techniques: Control, Staining

Journal: Redox Biology

Article Title: Melatonin protects aged oocytes from depalmitoylation-mediated quality reduction by promoting PPT1 degradation and antioxidation

doi: 10.1016/j.redox.2025.103510

Figure Lengend Snippet: Maternal age-associated oocyte spindle and chromosome abnormalities were suppressed by PPT1 knockdown and tubulin was detected to bind PPT1 by mass spectrometry. (A) The relative mRNA level of PPT1 in control and siPPT1 oocytes was determined by quantitative RT-PCR. (B) Western blot analysis showing PPT1 expression in control and siPPT1-treated oocytes. (C) Analysis of the palmitoylation levels of proteins extracted from aged and aged + siPPT1 oocytes. (D) Oocytes in the control, aged and aged + siPPT1 groups were collected and stained to visualize spindles (green) and chromosomes (red). Scale bar, 20 μm. (E) Quantitative analysis of oocytes in the control (n = 116), aged (n = 116) and aged + siPPT1 (n = 117) groups with spindle defects or chromosome misalignment. (F) Quantitative analysis of spindle width in control (n = 30), aged (n = 36) and aged + siPPT1 (n = 30) oocytes. (G) Schematic of mass spectrometry detection of PPT1-binding proteins. (H) Analysis of the binding of PPT1 to tubulin alpha 1A, tubulin alpha 1B and tubulin beta 5 by mass spectrometry. (I, J) Representative spectrum for the tubulin beta 5 peptide. (K) Schematic representation of tubulin palmitoylation sites identified by mass spectrometry. Actin served as a loading control for Western blotting and ABE assay. The data are presented as the means ± SDs from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The membranes were blocked with 5 % BSA at room temperature for 1 h and incubated with the following primary antibodies diluted to the appropriate concentration in a shaker at 4 °C overnight: anti-PPT1 antibody (1:3000), anti-actin antibody (Proteintech, 66009-1-Ig; 1:5000) and anti-tubulin antibody (Cell Signaling Technology, 2148s, 1:5000).

Techniques: Knockdown, Mass Spectrometry, Control, Quantitative RT-PCR, Western Blot, Expressing, Staining, Binding Assay

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: 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 in individual glomerulus was quantified ( n = 40 glomeruli in B6; n = 42 glomeruli in Ppt1 +/+ B6. Sle1yaa and Ppt1 -/- B6. Sle1yaa ). G Splenic CD11b + cell distribution is indicated by FACS plots (left), percentages (middle), and cell numbers (right). H Splenic CD4 + T cell activation is indicated by FACS plots (left), percentages (middle), and cell numbers (right). I Splenic CD8 + T cell activation is indicated by FACS plots (left), percentages (middle), and cell numbers (right). J Splenic plasma cell distribution is indicated by FACS plots (left), percentages (middle), and cell numbers (right). All data are pooled from three independent experiments (mean ± SEM.; P values were calculated by two-way Student’s t test).

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 Abcam goat anti-mouse IgG H&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, Immunofluorescence, Activation Assay

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: A pDCs enriched from PBMCs of healthy volunteers were treated with DMSO or HDSF overnight. After CpG A stimulation, IFNα levels was evaluated by ELISAs ( n = 9 individuals). B Sorted Flt3L-pDCs were treated with CpG A at the indicated timepoints. Ppt1 (left) and Ifna (right) qPCR results are shown ( n = 3 mice per group). C Sorted Ppt1 +/+ or Ppt1 -/- BM pDCs were treated with TLR9/TLR7 agonizts (left). CpG A and R848 ELISA results are shown ( n = 4 mice per group). D Ppt1 +/+ or Ppt1 -/- mice were infected with HSV (left). Serum IFNα ELISAs for HSV (right) are shown ( n = 5 mice for HSV). E Ppt1 +/+ or Ppt1 -/- indicated immune cells were treated with CpG B treatment. TNF ELISA results for CD19 + B cells, Per.MΦ and BMDMs are shown ( n = 4 mice per group for CD19 + B cells and Per.MΦ, n = 3 mice per group for BMDMs). F WT indicated immune cells were treated with DMSO or HDSF, followed by a CpG A (pDCs) or CpG B (B cells and macrophages) pulse. TNF ELISA results for sorted BM pDCs, CD19 + B cells, Per.MΦ and BMDMs are shown ( n = 4 mice per group for sorted BM pDCs, CD19 + B cells and Per.MΦ, n = 3 mice per group for BMDMs). All data are representative of three (mean ± SEM.; P values were calculated by two-way Student’s t test), except for those presented in ( A ), which were pooled from three independent experiments ( P values were calculated by two-tailed Wilcoxon matched-pair signed rank test).

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 Abcam goat anti-mouse IgG H&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: Enzyme-linked Immunosorbent Assay, Infection, Two Tailed Test

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: 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). D ABE assays were performed on 293 T cells transfected with PPT1 and mTLR7-FLAG ( n = 3 replicates). E ABE assays were performed on Ppt1 +/+ or Ppt1 -/- Flt3L-pDCs after 4 h (top) and 24 h (bottom) of CpG A treatment ( n = 3 replicates). F ABE assays were performed on Ppt1 +/+ or Ppt1 -/- RAW264.7 cells transduced with mTLR9-HA after 4 h of CpG A treatment ( n = 3 replicates). G RAW264.7 cells transduced with mTLR9-HA were treated with DMSO or HDSF overnight. ABE assays were performed after the addition of CpG A ( n = 3 replicates). All data are pooled from three independent experiments (mean ± SEM. P values were calculated by two-way Student’s t test).

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 Abcam goat anti-mouse IgG H&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: A TNF production 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, calculated 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 -/- RAW264.7 cells transduced with mTLR9-HA ( n = 4 replicates). F Immunoprecipitation assays with CpG B-biotin were performed on Ppt1 +/+ or Ppt1 -/- RAW264.7 cells transduced with mTLR9-HA ( n = 3 replicates). G RAW264.7 cells were treated with 2-BP or HDSF overnight. TNF production was measured by intracellular staining after addition of CpG B ( n = 4 replicates). H Immunoprecipitation assays with CpG B-biotin were performed on RAW264.7 cells treated with 2-BP or HDSF ( n = 3 replicates). I Cell fractionation of Zdhhc3 +/+ or Zdhhc3 -/- RAW264.7 cells transduced with mTLR9-HA showing the distributions of TLR9 in the indicated organelle markers. A representative blot (left) and the ratio of the full length mTLR9 in Golgi (right, quantified as the ratio of boxed fractions to all fractions, n = 3 replicates). J Immunoprecipitation of TLR9-HA from the DMSO or HDSF treated RAW macrophage lines in a followed by immunoblot of UNC93B1-FLAG. UNC93B1 levels in whole-cell lysates are also shown. A representative blot (left) and the ratio of the IP UNC93B1-FLAG in HDSF treated cells to the IP UNC93B1-FLAG in DMSO treated cells, n = 3 replicates. All data are representative of three independent experiment (mean ± SEM. P values were calculated by two-way Student’s t test).

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 Abcam goat anti-mouse IgG H&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, Transduction, Immunoprecipitation, Ligand Binding Assay, Cell Fractionation, Western Blot

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: 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-thiopalmitoyl-glucoside (Moscerdam, EM06650), Z-VAD-FMK (Bio Techne, FMK001), sorafenib (Santa Cruz Biotechnology, Sc-357,801), hexadecanesulfonyl fluoride (HDSF; Santa Cruz Biotechnology, sc-221,708), human PPT1 (OriGene Technologies, TP721098), DC661 (Vagdevi Innoscience), Triton X-100 (Dutscher, 091584B) and cOmplete™ Protease Inhibitor Cocktail (Roche, 4,693,132,001) were used.

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

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: 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 Zn 2+ 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-thiopalmitoyl-glucoside (Moscerdam, EM06650), Z-VAD-FMK (Bio Techne, FMK001), sorafenib (Santa Cruz Biotechnology, Sc-357,801), hexadecanesulfonyl fluoride (HDSF; Santa Cruz Biotechnology, sc-221,708), human PPT1 (OriGene Technologies, TP721098), DC661 (Vagdevi Innoscience), Triton X-100 (Dutscher, 091584B) and cOmplete™ Protease Inhibitor Cocktail (Roche, 4,693,132,001) were used.

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