full length ppt1 plasmid  (Addgene inc)

Journal: Cancer discovery

Article Title: PPT1 promotes tumor growth and is the molecular target of chloroquine derivatives in cancer.

doi: 10.1158/2159-8290.CD-18-0706

Figure Lengend Snippet: PPT1 is the unified target of CQ-derivatives. (A) A375P cells were treated with HCQ, Lys05 or DC661 (3 μM, 6 hrs) in media with a pH of 7.4, 6.5, or 6, and lysate was subsequently immunoblotted. (B) A375P cells were treated in an MTT assay with HCQ, Lys05 or DC661 (0 – 3 μM, 72 hrs) in media with a pH of 7.4, 6.5, or 6. (C) Chemical structure of the DC661-photoprobe (DC661-P). Red signifies the chemical warhead, green signifies the photoaffinity label, and blue signifies the desthiobiotin probe. (D) Proteomic results of eluents generated from A375P cells treated with either DC661-P (400 nM) minus UV, DC661-P plus UV, or DC661-P plus UV and plus DC661 competition (4 μM, 1 hr). (E) Lysate from A375P cells treated as described from figure 1D was immunoblotted for PPT1. (F) Chemical structure of the Lys05-photoprobe (Lys05-P). (G) Lysate generated from A375P cells treated with either Lys05-P (400 nM) minus UV, Lys05-P plus UV, or Lys05-P plus UV and plus Lys05 competition (40 μM, 1 hr) was immunoblotted for PPT1. (H) Chemical structure of the CQ-photoprobe (CQ-P). (I) Lysate generated from A375P cells treated with CQ-P (400 nM) minus UV, CQ-P plus UV, or CQ-P plus UV and plus HCQ competition (40 μM, 1 hr) was immunoblotted for PPT1. (J) Differential scanning calorimetry was performed using HCQ+PPT1, Lys05+PPT1, and DC661+PPT1 against apo-PPT1 ligand. (K) PPT1 enzymatic assays were performed in A375P cells treated with HCQ, Lys05, or DC661 (0 – 100 μM, 1 hr). (L) Acyl biotin exchange assays were performed in A375P cells treated with HCQ or DC661 (3 μM, 1 hr). All data is representative of at least 2 experiments. Standard deviation is shown in (B), (J) and (K). * indicates p<0.05. Students T test was used.

Article Snippet: Full length PPT1 plasmid was obtained from Addgene (Plasmid #25205).

Techniques: MTT Assay, Generated, Differential Scanning Calorimetry, Standard Deviation

Journal: Cancer discovery

Article Title: PPT1 promotes tumor growth and is the molecular target of chloroquine derivatives in cancer.

doi: 10.1158/2159-8290.CD-18-0706

Figure Lengend Snippet: PPT1 inhibition leads to lysosomal inhibition. (A) A375P cells expressing the mCherry-eGFP-LC3B reporter were treated with HCQ (3 μM, 1 hr), Lys05, (3 μM, 1 hr), or DC661 (3 μM, 1 hr) in the presence or absence of N-tert-Butylhydroxylamine (NTBHA, 2 mM), and subsequently analyzed by microscopy. Shown are quantitation. (B) A375P WT PPT1 or KO PPT1 cells were treated with HDSF (60 μM), HCQ (3 μM), Lys05 (3 μM) or DC661 (3 μM) for 6 hours before being stained with Lysosensor. To the right is quantitation of Lysosensor intensity. (C) A375P WT PPT1 or KO PPT1 cells were treated with HCQ, Lys05, or DC661 (3 μM, 0 – 6 hr). Lysate was immunoblotted (D) A375P WT PPT1 or KO PPT1 cells were treated with HCQ (30 μM), Lys05 (3 μM), or DC661 (1 μM, 72 hrs) and cell numbers were quantified as shown. (E) B16 WT Ppt1 or KO Ppt1 cells were treated with HCQ, Lys05, or DC661 (3 μM, 1 hr). Lysate was immunoblotted. (F) Proximity ligation assay for the p18 (Ragulator) – V1A (vATPase subunit) interaction in WT PPT1 and KO PPT1 cells. Shown below is quantitation. (G) Proximity ligation assay for the mTOR – Rheb interaction in WT PPT1 and KO PPT1 cells. Shown below is quantitation. (H) Membrane fractions generated from WT PPT1 and KO PPT1 cells were subsequently immunoblotted. All data is representative of at least 2 experiments. Standard deviation is shown in (D), (F), and (G). * indicates p<0.05. Students T test was used.

Article Snippet: Full length PPT1 plasmid was obtained from Addgene (Plasmid #25205).

Techniques: Inhibition, Expressing, Microscopy, Quantitation Assay, Staining, Proximity Ligation Assay, Membrane, Generated, Standard Deviation

Journal: Cancer discovery

Article Title: PPT1 promotes tumor growth and is the molecular target of chloroquine derivatives in cancer.

doi: 10.1158/2159-8290.CD-18-0706

Figure Lengend Snippet: PPT1 expression is correlated with poorer patient survival. (A) WT PPT1 or KO PPT1 cells (1 × 106 cells/mouse were injected into the flanks of NSG mice (n=5, each arm). Tumor volumes were measured every three days as shown. (B) Mean +/− SEM daily tumor growth rate. (C) Lysate generated from WT PPT1 or KO PPT1 cells grown in vivo were immunoblotted. (D) PPT1 expression (TCGA) in unpaired normal (symbol: triangles) and tumor samples (symbol: stars) for the following cancers: BRCA: breast cancer; KIRC: clear cell renal cell carcinoma; THCA: thyroid cancer; HNSC: head and neck squamous cell carcinoma; ESCA: esophageal cancer; STAD: gastric cancer; LIHC: hepatocellular carcinoma; KIRP: Papillary renal cell carcinoma; COAD: colon cancer; BLCA: bladder cancer; PRAD: prostate adenocarcinoma; LUSC: non-small cell lung cancer squamous cell; LUAD: non-small cell lung cancer adenocarcinoma. Mean +/− SEM is presented; *p<0.05 paired t-test. (E) PPT1 expression (TCGA RNAseqV2) in primary versus metastatic melanoma tumor samples. PPT1 values less than the 30th percentile were classified as low and values greater than the 70th percentile were classified as low. (F-I) Kaplan-Meier survival curves were computed for medium/high versus low or high versus medium/low expression of PPT1 in TCGA patients with complete RNASeqV2 expression data as well as overall survival information. Hazard ratio with 95% confidence interval and associated p-value are from a Cox regression analysis. (F) esophageal cancer, (G) hepatocellular carcinoma, (H) clear cell renal cell carcinoma, (I) head and neck cancer. Low, medium and high was defined based on tertiles for PPT1.

Article Snippet: Full length PPT1 plasmid was obtained from Addgene (Plasmid #25205).

Techniques: Expressing, Injection, Generated, In Vivo

Journal: Cancer discovery

Article Title: PPT1 promotes tumor growth and is the molecular target of chloroquine derivatives in cancer.

doi: 10.1158/2159-8290.CD-18-0706

Figure Lengend Snippet: PPT1 is the unified target of CQ-derivatives. (A) A375P cells were treated with HCQ, Lys05 or DC661 (3 μM, 6 hrs) in media with a pH of 7.4, 6.5, or 6, and lysate was subsequently immunoblotted. (B) A375P cells were treated in an MTT assay with HCQ, Lys05 or DC661 (0 – 3 μM, 72 hrs) in media with a pH of 7.4, 6.5, or 6. (C) Chemical structure of the DC661-photoprobe (DC661-P). Red signifies the chemical warhead, green signifies the photoaffinity label, and blue signifies the desthiobiotin probe. (D) Proteomic results of eluents generated from A375P cells treated with either DC661-P (400 nM) minus UV, DC661-P plus UV, or DC661-P plus UV and plus DC661 competition (4 μM, 1 hr). (E) Lysate from A375P cells treated as described from figure 1D was immunoblotted for PPT1. (F) Chemical structure of the Lys05-photoprobe (Lys05-P). (G) Lysate generated from A375P cells treated with either Lys05-P (400 nM) minus UV, Lys05-P plus UV, or Lys05-P plus UV and plus Lys05 competition (40 μM, 1 hr) was immunoblotted for PPT1. (H) Chemical structure of the CQ-photoprobe (CQ-P). (I) Lysate generated from A375P cells treated with CQ-P (400 nM) minus UV, CQ-P plus UV, or CQ-P plus UV and plus HCQ competition (40 μM, 1 hr) was immunoblotted for PPT1. (J) Differential scanning calorimetry was performed using HCQ+PPT1, Lys05+PPT1, and DC661+PPT1 against apo-PPT1 ligand. (K) PPT1 enzymatic assays were performed in A375P cells treated with HCQ, Lys05, or DC661 (0 – 100 μM, 1 hr). (L) Acyl biotin exchange assays were performed in A375P cells treated with HCQ or DC661 (3 μM, 1 hr). All data is representative of at least 2 experiments. Standard deviation is shown in (B), (J) and (K). * indicates p<0.05. Students T test was used.

Article Snippet: Proteomics DC661-P pulldowns from cell lysate were performed as previously described ( 9 ) Purification of PPT1 Full length PPT1 plasmid was obtained from Addgene (Plasmid #25205).

Techniques: MTT Assay, Generated, Differential Scanning Calorimetry, Standard Deviation

Journal: Cancer discovery

Article Title: PPT1 promotes tumor growth and is the molecular target of chloroquine derivatives in cancer.

doi: 10.1158/2159-8290.CD-18-0706

Figure Lengend Snippet: PPT1 inhibition leads to lysosomal inhibition. (A) A375P cells expressing the mCherry-eGFP-LC3B reporter were treated with HCQ (3 μM, 1 hr), Lys05, (3 μM, 1 hr), or DC661 (3 μM, 1 hr) in the presence or absence of N-tert-Butylhydroxylamine (NTBHA, 2 mM), and subsequently analyzed by microscopy. Shown are quantitation. (B) A375P WT PPT1 or KO PPT1 cells were treated with HDSF (60 μM), HCQ (3 μM), Lys05 (3 μM) or DC661 (3 μM) for 6 hours before being stained with Lysosensor. To the right is quantitation of Lysosensor intensity. (C) A375P WT PPT1 or KO PPT1 cells were treated with HCQ, Lys05, or DC661 (3 μM, 0 – 6 hr). Lysate was immunoblotted (D) A375P WT PPT1 or KO PPT1 cells were treated with HCQ (30 μM), Lys05 (3 μM), or DC661 (1 μM, 72 hrs) and cell numbers were quantified as shown. (E) B16 WT Ppt1 or KO Ppt1 cells were treated with HCQ, Lys05, or DC661 (3 μM, 1 hr). Lysate was immunoblotted. (F) Proximity ligation assay for the p18 (Ragulator) – V1A (vATPase subunit) interaction in WT PPT1 and KO PPT1 cells. Shown below is quantitation. (G) Proximity ligation assay for the mTOR – Rheb interaction in WT PPT1 and KO PPT1 cells. Shown below is quantitation. (H) Membrane fractions generated from WT PPT1 and KO PPT1 cells were subsequently immunoblotted. All data is representative of at least 2 experiments. Standard deviation is shown in (D), (F), and (G). * indicates p<0.05. Students T test was used.

Article Snippet: Proteomics DC661-P pulldowns from cell lysate were performed as previously described ( 9 ) Purification of PPT1 Full length PPT1 plasmid was obtained from Addgene (Plasmid #25205).

Techniques: Inhibition, Expressing, Microscopy, Quantitation Assay, Staining, Proximity Ligation Assay, Membrane, Generated, Standard Deviation

Journal: Cancer discovery

Article Title: PPT1 promotes tumor growth and is the molecular target of chloroquine derivatives in cancer.

doi: 10.1158/2159-8290.CD-18-0706

Figure Lengend Snippet: PPT1 expression is correlated with poorer patient survival. (A) WT PPT1 or KO PPT1 cells (1 × 106 cells/mouse were injected into the flanks of NSG mice (n=5, each arm). Tumor volumes were measured every three days as shown. (B) Mean +/− SEM daily tumor growth rate. (C) Lysate generated from WT PPT1 or KO PPT1 cells grown in vivo were immunoblotted. (D) PPT1 expression (TCGA) in unpaired normal (symbol: triangles) and tumor samples (symbol: stars) for the following cancers: BRCA: breast cancer; KIRC: clear cell renal cell carcinoma; THCA: thyroid cancer; HNSC: head and neck squamous cell carcinoma; ESCA: esophageal cancer; STAD: gastric cancer; LIHC: hepatocellular carcinoma; KIRP: Papillary renal cell carcinoma; COAD: colon cancer; BLCA: bladder cancer; PRAD: prostate adenocarcinoma; LUSC: non-small cell lung cancer squamous cell; LUAD: non-small cell lung cancer adenocarcinoma. Mean +/− SEM is presented; *p<0.05 paired t-test. (E) PPT1 expression (TCGA RNAseqV2) in primary versus metastatic melanoma tumor samples. PPT1 values less than the 30th percentile were classified as low and values greater than the 70th percentile were classified as low. (F-I) Kaplan-Meier survival curves were computed for medium/high versus low or high versus medium/low expression of PPT1 in TCGA patients with complete RNASeqV2 expression data as well as overall survival information. Hazard ratio with 95% confidence interval and associated p-value are from a Cox regression analysis. (F) esophageal cancer, (G) hepatocellular carcinoma, (H) clear cell renal cell carcinoma, (I) head and neck cancer. Low, medium and high was defined based on tertiles for PPT1.

Article Snippet: Proteomics DC661-P pulldowns from cell lysate were performed as previously described ( 9 ) Purification of PPT1 Full length PPT1 plasmid was obtained from Addgene (Plasmid #25205).

Techniques: Expressing, Injection, Generated, In Vivo