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: 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

Journal: Advanced Science

Article Title: PIK3CA Mutations Downregulate PPT1 to Promote Adipogenesis by Suppressing P300 Depalmitoylation and Phase Separation

doi: 10.1002/advs.202523139

Figure Lengend Snippet: PPT1 physically interacted with P300. (A) Gel electrophoresis was performed after using PPT1 antibody for PPT1‐immunoprecipitation, then Coomassie brilliant blue staining was conducted. The red frame showed the PPT1 and P300 predicted based on molecular weight. (B) The results of immunoprecipitation‐mass spectrometry (IP‐MS) analysis in Im FIL‐ASPCs with or without PPT1 overexpression. (C) Mass spectrometry analysis of a peptide derived from PPT1‐immunoprecipitates to show the potential interaction between PPT1 and P300. (D) The predicted model of the protein complex was obtained through computation via the AlphaFold3 server. The output file was further analyzed with PyMOL, and detailed images of the binding interface between the two proteins were generated. (E) PPT1 and P300 were detected by immunofluorescence staining in FIL‐ASPCs and Im FIL‐ASPCs (left). The co‐localization analysis was performed by Image J (right). The red line indicated the region used for measuring the colocalization signal. (F) Proximity ligation assays (PLA) were performed using anti‐PPT1 and anti‐P300 antibodies in FIL‐ASPCs and Im FIL‐ASPCs. Red signals represent PLA signals, indicating the proximity of the targeted proteins, while blue signals denote the cell nuclei. (G, H) FIL‐ASPCs cellular lysates were analyzed by Co‐IP followed by western blotting. (I) Schematic representation of PPT1 and P300 truncations. (J) IP and western blot assay of the interaction between FLAG‐tagged truncated PPT1 and Myc‐tagged P300 in HEK293T cells. Cell extracts were IP with an anti‐Flag antibody. (K) IP and western blot assay of the interaction between FLAG‐tagged PPT1 and Myc‐tagged truncated P300 in HEK293T cells. Cell extracts were IP with an anti‐Myc antibody. (L) IP and western blot assay show the interactions between FLAG‐tagged mutated PPT1 and Myc‐tagged mutated P300 in HEK293T cells. The point mutations were designed according to the scheme showed in Figure .

Article Snippet: The primary antibodies used were as follows: anti‐PPT1 (Proteintech, 29653‐1‐AP), anti‐p110α (Proteintech, 67071‐1‐Ig), anti‐AKT (Cell Signaling Technology, 4691), anti‐phospho‐AKT (Cell Signaling Technology, 9271), anti‐JUN (Proteintech, 24909‐1‐AP), and anti‐phospho‐JUN (Proteintech, 28891‐1‐AP), anti‐PPAR γ (Cell Signaling Technology, 2443S), anti‐C/EBP α (Proteintech, 13274‐1‐AP), anti‐FABP 4 (Proteintech, 12802‐1‐AP), anti‐P300 (Proteintech, 20695‐1‐AP) anti‐H3K9ac (HUABIO, HA722132), anti‐H3K27ac (HUABIO, HA500046 ), anti‐Histone H4 (HUABIO, ET1612‐43), anti‐Flag (MERCK, F7425), anti‐Myc (Proteintech, 60003‐2‐Ig), anti‐VPS4A (Epizyme, 95M49L94), anti‐p62 (Epizyme, 78L28M94).

Techniques: Nucleic Acid Electrophoresis, Immunoprecipitation, Staining, Molecular Weight, Mass Spectrometry, Protein-Protein interactions, Over Expression, Derivative Assay, Binding Assay, Generated, Immunofluorescence, Ligation, Co-Immunoprecipitation Assay, Western Blot

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: 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