biotin dc puromycin jena bioscience cat  (Jena Bioscience)

Journal: Advanced Science

Article Title: m6Am Methyltransferase PCIF1 Promotes LPP3 Mediated Phosphatidic Acid Metabolism and Renal Cell Carcinoma Progression

doi: 10.1002/advs.202404033

Figure Lengend Snippet: PCIF1 expression is significantly increased in RCC and associated with poor prognosis. A) The expression of PCIF1 in kidney renal cell carcinoma (KIRC) was analyzed with the TCGA database. B) The qRT‐PCR assay results show PCIF1 expression at the mRNA level in RCC specimens and adjacent normal tissues from the SRRSH cohort. C,D) PCIF1 expression in normal tissues and RCC specimens in GSE14994 C) and GSE46699 D) datasets. Data are shown as the mean ± SD. E) The protein expression of PCIF1 in RCC specimens and adjacent normal tissues in the CPTAC database. F) The western blot assays exhibited the PCIF1 protein expression in paired RCC tumor and normal tissues from the SRRSH RCC cohort. G) Representative IHC staining images for PCIF1 protein in the SRRSH RCC cohort are presented. IHC scores are calculated and analyzed. H) Representative immunofluorescence images in Caki‐1 and OS‐RC‐2 cells stained with anti‐PCIF1. Nuclei were stained with DAPI. Scale bar, 10 µm. I) LC‐MS/MS quantification of the m6Am/A ratios in RNAs extracted from 8 pairs of RCC specimens and their corresponding normal tissues. J) Relative PCIF1 expression of SRRSH RCC cohort with or without metastasis. K) The frequency of relapse in RCC patients with low and high expression of PCIF1, the low and high PCIF1 expression groups were cut off by the median expression. L) Kaplan–Meier survival curves of low and high PCIF1 expression groups. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: 3 × 10 6 PCIF1 KD and control RCC cells were incubated with 1:1000 Biotin‐dC‐puromycin (NU‐925‐BIO‐S, Jena Bioscience) for 12 h. Cells were then collected and lysed in 1 mL lysis buffer (1% NP40, 20 m m Tris‐HCl, PH 7.4, 150 m m NaCl, 10% glycerol, protease inhibitor cocktail).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Immunofluorescence, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Advanced Science

Article Title: m6Am Methyltransferase PCIF1 Promotes LPP3 Mediated Phosphatidic Acid Metabolism and Renal Cell Carcinoma Progression

doi: 10.1002/advs.202404033

Figure Lengend Snippet: PCIF1 is required for RCC progression in vitro and in vivo. A) qRT‐PCR and western blotting confirmed the knockdown of PCIF1 in Caki‐1 and OS‐RC‐2 cells. B) Optical density at 450 nm (OD450) values of Caki‐1 cells transfected with control or PCIF1 siRNAs in CCK‐8 assay. C) Representative images of colony‐formation assay and its quantification data of indicated Caki‐1 cells. D) Flow cytometric analysis of cell cycle in Caki‐1 cells transfected with control or PCIF1 siRNAs. E) Representative images of EdU assay in indicated Caki‐1 cells. Scale bar, 100 µm. F) Representative images of transwell assay in indicated Caki‐1 cells. Scale bar, 100 µm. The migrated cells are counted and analyzed. G) qRT‐PCR and western blotting showing the overexpression of wild‐type or mutant PCIF1 (N553A) in Caki‐1 and OS‐RC‐2 cells. H) Optical density at 450 nm (OD450) values of Caki‐1 cells overexpressed with vector or wild‐type/mutant PCIF1 in CCK‐8 assay. I) Representative images of colony‐formation assay and its quantification data of indicated Caki‐1 cells. J) Flow cytometric analysis of cell cycle in Caki‐1 cells transfected with vector or wild‐type/mutant PCIF1. K) Representative images of EdU assay in indicated Caki‐1 cells. Scale bar, 100 µm. L) Representative images of transwell assay in indicated Caki‐1 cells. Scale bar, 100 µm. The migrated cells are counted and analyzed. M) Western blotting showing the PCIF1 depletion in OS‐RC‐2 cells with lentivirus‐based control or shRNA. N–P) Images N), volumes O), and weights P) of indicated OS‐RC‐2 cell‐derived xenograft tumors (n = 6). Q) Representative H&E and IHC staining images for PCIF1, PCNA of OS‐RC‐2 cell‐derived xenograft tumors. Scale bar, 100 µm. R) Western blotting showing the PCIF1 depletion in ACHN cells with lentivirus‐based control or shRNA. S,T) Bioluminescent images showing primary foci and metastasis in mice underwent luciferase labeled ACHN injection under renal capsule (n = 5) S). Bioluminescent signal intensities (photons/s/cm2/sr) of primary foci were quantified T). U) Representative bioluminescent images of metastases in the liver and intestine. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: 3 × 10 6 PCIF1 KD and control RCC cells were incubated with 1:1000 Biotin‐dC‐puromycin (NU‐925‐BIO‐S, Jena Bioscience) for 12 h. Cells were then collected and lysed in 1 mL lysis buffer (1% NP40, 20 m m Tris‐HCl, PH 7.4, 150 m m NaCl, 10% glycerol, protease inhibitor cocktail).

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Western Blot, Knockdown, Transfection, Control, CCK-8 Assay, Colony Assay, EdU Assay, Transwell Assay, Over Expression, Mutagenesis, Plasmid Preparation, shRNA, Derivative Assay, Immunohistochemistry, Luciferase, Labeling, Injection

Journal: Advanced Science

Article Title: m6Am Methyltransferase PCIF1 Promotes LPP3 Mediated Phosphatidic Acid Metabolism and Renal Cell Carcinoma Progression

doi: 10.1002/advs.202404033

Figure Lengend Snippet: LPP3 was identified as a downstream target of PCIF1. A) Metageneplot showing the distribution of m6Am peaks across mRNA. B) Consensus motif of m6Am peaks presented by HOMER. C) List of genes with differentially exhibited 5'‐UTR m6Am peaks upon PCIF1 knockdown in OS‐RC‐2 cells, log2FC < −0.5, p < 0.01. D) Heatmap showing the mRNA expression change in OS‐RC‐2 upon PCIF1 knockdown. E) Bubble diagram showing the biological processes enrichment of differentially expressed genes upon PCIF1 knockdown. F) Gene set enrichment analysis of Glycerophospholipid Catabolic Process pathway upon PCIF1 knockdown. G) Integrative Genomics Viewer (IGV) tracks displaying m6Am peaks on LPP3 in control and PCIF1‐depleted OS‐RC‐2 cells. H,I) m6A‐RIP assay showing the m6A/m6Am level on LPP3 in RCC cells with the silencing H) or overexpression I) of PCIF1. J) qRT‐PCR assay showing the mRNA level of LPP3 in RCC cells transfected with indicated PCIF1 siRNAs. K,L) Western blotting assay showing the protein level of LPP3 in RCC cells transfected with indicated PCIF1 siRNAs and plasmids. M) qRT‐PCR assay showing the distribution of LPP3 mRNA in different polysome gradient fractions in control and PCIF1‐depleted RCC cells. N) Western blotting assay showing the effect of PCIF1 knockdown on nascent LPP3 labeled with biotin‐dC‐puromycin. O) Western blotting showing the protein level of LPP3 in RCC cells transfected with indicated plasmids. P) Schematic diagram of LPP3 5′‐UTR WT and 5′‐UTR MUT firefly luciferase reporters. Relative luciferase activity in 293T cells transfected with firefly luciferase reporters, renilla luciferase vector, and indicated PCIF1 overexpression plasmids. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: 3 × 10 6 PCIF1 KD and control RCC cells were incubated with 1:1000 Biotin‐dC‐puromycin (NU‐925‐BIO‐S, Jena Bioscience) for 12 h. Cells were then collected and lysed in 1 mL lysis buffer (1% NP40, 20 m m Tris‐HCl, PH 7.4, 150 m m NaCl, 10% glycerol, protease inhibitor cocktail).

Techniques: Knockdown, Expressing, Control, Over Expression, Quantitative RT-PCR, Transfection, Western Blot, Labeling, Luciferase, Activity Assay, Plasmid Preparation

Journal: Advanced Science

Article Title: m6Am Methyltransferase PCIF1 Promotes LPP3 Mediated Phosphatidic Acid Metabolism and Renal Cell Carcinoma Progression

doi: 10.1002/advs.202404033

Figure Lengend Snippet: LPP3 mediated the oncogenic role of PCIF1 in RCC progression. A) The protein expression of LPP3 in RCC specimens and adjacent normal tissues in the CPTAC database. B) The western blotting shows the LPP3 protein expression in paired RCC tumor and normal tissues from the SRRSH RCC cohort. C) Representative IHC staining images for LPP3 protein in the SRRSH RCC cohort. Scale bar, 100 µm. IHC scores are calculated and analyzed. D,E) Pearson's correlation analysis shows a positive correlation between PCIF1 and LPP3 expression in proteomics data from the CPTAC RCC dataset (D, n = 110) and IHC scores of the SRRSH RCC cohort (E, n = 30). F) Representative IHC staining images for PCIF1 and LPP3 protein of OS‐RC‐2 cell‐derived xenograft tumors. Scale bar, 100 µm. IHC scores are calculated and analyzed. G) Western blotting showing the protein level of LPP3 in RCC cells transfected with indicated plasmids and siRNAs. H) Optical density at 450 nm (OD450) values of OS‐RC‐2 cells transfected with indicated plasmids and siRNAs in CCK‐8 assay. I) Representative images of colony‐formation assay and its quantification data of indicated OS‐RC‐2 cells. J) Representative images of EdU assay of indicated OS‐RC‐2 cells. Scale bar, 100 µm. K) Representative images of transwell assay in OS‐RC‐2 cells transfected indicated plasmids and siRNAs. Scale bar, 100 µm. The migrated cells are counted and analyzed. L) Optical density at 450 nm (OD450) values of OS‐RC‐2 cells treated with vehicle or XY‐14 (10µM) in cell counting kit‐8 (CCK‐8) assay. M) Representative images of colony‐formation assay and its quantification data of OS‐RC‐2 cells treated with vehicle or XY‐14 (10 µM). N) Representative images of transwell assay in OS‐RC‐2 cells treated with vehicle or XY‐14 (10 µM). Scale bar, 100 µm. The migrated cells are counted and analyzed. (O‐Q) Images O), volumes P), and weights Q) of cell‐derived tumors from control or PCIF1 overexpressed OS‐RC‐2 cells treated with vehicle or XY‐14 (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: 3 × 10 6 PCIF1 KD and control RCC cells were incubated with 1:1000 Biotin‐dC‐puromycin (NU‐925‐BIO‐S, Jena Bioscience) for 12 h. Cells were then collected and lysed in 1 mL lysis buffer (1% NP40, 20 m m Tris‐HCl, PH 7.4, 150 m m NaCl, 10% glycerol, protease inhibitor cocktail).

Techniques: Expressing, Western Blot, Immunohistochemistry, Derivative Assay, Transfection, CCK-8 Assay, Colony Assay, EdU Assay, Transwell Assay, Cell Counting, Control

Journal: Advanced Science

Article Title: m6Am Methyltransferase PCIF1 Promotes LPP3 Mediated Phosphatidic Acid Metabolism and Renal Cell Carcinoma Progression

doi: 10.1002/advs.202404033

Figure Lengend Snippet: LPP3 knockdown led to phosphatidic acid piling up in mitochondria. A) Schematic diagram showing LPP3 catalyzes the hydrolysis of phosphatidic acid. B) Representative immunofluorescence images in Caki‐1 and OS‐RC‐2 cells stained with anti‐LPP3, anti‐GRP78, and DAPI. Scale bar, 10 µm. C) Western blotting of LPP3 and different markers in the endoplasmic reticulum and mitochondria fractions of RCC cells. D) Correlation of LPP3 expression and KEGG Glycerophospholipid Metabolism enrichment scores in malignant cells in two RCC tumor single‐cell datasets GSE156632 and GSE159115. (E‐G) The Elisa assay shows the level of cellular E), endoplasmic reticular F), and mitochondrial G) PA. Data are representative of three independent experiments and are shown as the mean ± SD. H) Quantification of mitochondrial PA level of control and PCIF1‐knockdown Caki‐1 cells, relative to control condition. I) Quantification of different PA subclasses levels in mitochondria of control and PCIF1‐knockdown Caki‐1 cells, relative to control condition. Data are representative of two independent experiments and are shown as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: 3 × 10 6 PCIF1 KD and control RCC cells were incubated with 1:1000 Biotin‐dC‐puromycin (NU‐925‐BIO‐S, Jena Bioscience) for 12 h. Cells were then collected and lysed in 1 mL lysis buffer (1% NP40, 20 m m Tris‐HCl, PH 7.4, 150 m m NaCl, 10% glycerol, protease inhibitor cocktail).

Techniques: Knockdown, Immunofluorescence, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Advanced Science

Article Title: m6Am Methyltransferase PCIF1 Promotes LPP3 Mediated Phosphatidic Acid Metabolism and Renal Cell Carcinoma Progression

doi: 10.1002/advs.202404033

Figure Lengend Snippet: PCIF1/LPP3 axis facilitated mitochondrial fission and enhanced mitochondrial respiration in RCC. A) Representative morphology of mitochondria stained by mitotracker in indicated Caki‐1 cells and quantification of mitochondrial network. Scale bar, 10 µm. Around 50 cells per group were assessed. B) Representative morphology of mitochondria revealed by electron microscopy in indicated Caki‐1 cells. Scale bar, 1 µm. C) Representative morphology of mitochondria stained by mitotracker in Caki‐1 cells transfected with indicated siRNAs and quantification of mitochondrial network. Scale bar, 10 µm. Around 50 cells per group were assessed. D) Western blotting showing the monomers and oligomers of DRP‐1 in Caki‐1 cells transfected with indicated siRNAs. E) Representative morphology of mitochondria stained by mitotracker in indicated Caki‐1 cells with quantification of mitochondrial network. Scale bar, 10 µm. Around 50 cells per group were assessed. F) Representative morphology of mitochondria revealed by electron microscopy in indicated Caki‐1 cells. Scale bar, 1 µm. G) Cellular ATP levels in indicated Caki‐1 cells. H) Flow cytometric analysis showing ROS levels in indicated Caki‐1 cells. The average ROS levels are calculated. I) Flow cytometric analysis showing the mitochondrial membrane potential in indicated Caki‐1 cells. The proportion of cells with JC‐1 monomers was quantified. J,K) Oxygen consumption rate was detected in indicated Caki‐1 cells. Oligomycin, FCCP, rotenone, and antimycin A were added at indicated time points. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant. Data are representative of three independent experiments and are shown as the mean ± SD.

Article Snippet: 3 × 10 6 PCIF1 KD and control RCC cells were incubated with 1:1000 Biotin‐dC‐puromycin (NU‐925‐BIO‐S, Jena Bioscience) for 12 h. Cells were then collected and lysed in 1 mL lysis buffer (1% NP40, 20 m m Tris‐HCl, PH 7.4, 150 m m NaCl, 10% glycerol, protease inhibitor cocktail).

Techniques: Staining, Electron Microscopy, Transfection, Western Blot, Membrane

Journal: Advanced Science

Article Title: m6Am Methyltransferase PCIF1 Promotes LPP3 Mediated Phosphatidic Acid Metabolism and Renal Cell Carcinoma Progression

doi: 10.1002/advs.202404033

Figure Lengend Snippet: Targeting PCIF1 sensitizes RCC to sunitinib treatment. A) Flow cytometric analysis showing the apoptotic ratio of Caki‐1 and OS‐RC‐2 cells with PCIF1 knockdown under 4 µM sunitinib treatment. B) CCK‐8 assay of control and PCIF1‐knockdown RCC cell lines with sunitinib treatment at indicated concentrations for 48 h. C–E) Volumes C), images D), and weights E) of cell‐derived tumors from control or PCIF1‐knockdown OS‐RC‐2 cells treated with DMSO or sunitinib (40 mg kg −1 day −1 ) (n = 7). F) Representative IHC staining images for PCNA, CD31 of OS‐RC‐2 cell‐derived xenograft tumors. Scale bar, 100 µm. IHC score and CD31 positive area are calculated and analyzed (n = 5). G) Diagram of the proposed molecular mechanisms for PCIF1 involvement in RCC progression. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: 3 × 10 6 PCIF1 KD and control RCC cells were incubated with 1:1000 Biotin‐dC‐puromycin (NU‐925‐BIO‐S, Jena Bioscience) for 12 h. Cells were then collected and lysed in 1 mL lysis buffer (1% NP40, 20 m m Tris‐HCl, PH 7.4, 150 m m NaCl, 10% glycerol, protease inhibitor cocktail).

Techniques: Knockdown, CCK-8 Assay, Control, Derivative Assay, Immunohistochemistry