Journal: Cancer Research

Article Title: PABPC1L Induces IDO1 to Promote Tryptophan Metabolism and Immune Suppression in Renal Cell Carcinoma

doi: 10.1158/0008-5472.CAN-23-2521

Figure Lengend Snippet: PABPC1L deficiency improves antitumor immunity in murine and human RCC. A and D, Schematic of Renca cells with/without PABPC1L knockdown subcutaneously injected into BALB/c ( A ) and nude ( D ) mice. B and C, Images of tumors ( B ) and growth curves ( C ) in BALB/c mice ( n = 5/group). E and F, Tumor images ( E ) and growth curves ( F ) in nude mice ( n = 5/group). G, Schematic of the experimental setup for the 3D collagen-fibrin gel killing assay (left) and quantification of target cell killing by tumor-isolated CD8 + T cells or tumor antigen–specific CD8 + T cells (right). H, Flow cytometry analysis of GZMB in CD8 + T cells isolated from indicated Renca tumors in BALB/c mice. I, Flow cytometric analysis of Foxp3 expression in tumor-infiltrating CD4 + populations isolated from indicated Renca tumors in BALB/c mice. J, In vitro suppressive activity of tumor-isolated Tregs isolated from shPABPC1L or shCtrl mice. Left, representative histograms of CD8 + T-cell proliferation. Right, FACS quantification of T-cell proliferation. K and L, Survival curves of BALB/c ( K ) and nude ( L ) mice with Renca cells ( n = 10/group). M, Flow cytometry analysis of HLA-A2 expression in primary kidney tumor cells. N, Representative flow cytometry histograms (left) and statistical analysis (right) of apoptosis rate (PI+) of primary kidney tumor cells with or without PABPC1L knockdown cocultured with primary kidney tumor-specific CD8 + T cells. O, The proportion of IFNγ-producing T cells in shPABPC1L or shCtrl samples. A – F, Data represent one independent experiment with 5 mice per group. G, Data are representative of five independent experiments. H – J, Each experiment was repeated three times with 5 mice per group, and data shown are the representative group of three independent experiments. K and L , Data represent one independent experiment with 10 mice per group. N and O , Data represent five independent biological replicates. ***, P < 0.001.

Article Snippet: The immortalized renal epithelial cell line (HK-2, RRID:CVCL_YE28), the human RCC cell lines [786-O (RRID: CVCL_1051), 769-P (RRID: CVCL_1050), A-498 (RRID:CVCL_1056), ACHN (RRID: CVCL_1067), and Caki-1 (RRID:CVCL_0234)] and the mouse RCC cell line (Renca, RRID:CVCL_2174) were purchased from ATCC.

Techniques: Knockdown, Injection, Isolation, Flow Cytometry, Expressing, In Vitro, Activity Assay

Journal: Cancer Research

Article Title: PABPC1L Induces IDO1 to Promote Tryptophan Metabolism and Immune Suppression in Renal Cell Carcinoma

doi: 10.1158/0008-5472.CAN-23-2521

Figure Lengend Snippet: Loss of PABPC1L impairs JAK-STAT-IDO1 pathway in RCC. A, Representative Western blot (top) and statistical analysis (bottom) in the immortalized HK-2 renal epithelial cell line and RCC cell lines. GAPDH was used as a loading control. B, KEGG analysis showed the significantly altered signaling pathways after PABPC1L silencing in RCC cells. C, Representative Western blot analysis of STAT1, STAT3, pSTAT1, and pSTAT3 protein expression levels in 769-P cells with PABPC1L knockdown (left) and in Caki-1 cells with PABPC1L overexpression (right). D, mRNA expression of JAK-STAT1 target genes in shCtrl and shPABPC1L 769-P cells with or without IFNγ stimulation. Cells were treated with 100 U/mL IFNγ. E, Representative Western blot analysis showing JAK1, p-JAK1, JAK2, p-JAK2, STAT1, pSTAT1, and IDO1 protein expression levels in 769-P (left) and Caki-1 (right) cells with PABPC1L knockdown. Cells were stimulated with or without 100 U/mL IFNγ. F, Effects of JAK2 overexpression on IDO1 protein expression in shCtrl and shPABPC1L 769-P cells. G, Effects of JAK2 overexpression on IDO1 mRNA expression in shCtrl and shPABPC1L 769-P cells. A – G, All experiments were performed with three independent biological replicates, and data shown are representative of three independent experiments. H, Differently treated cells were cultured for 24 hours without KYNU treatment. KYN and TRP levels in cell supernatants were determined by ELISA. Differently treated cells were injected subcutaneously into BALB/c mice. HPLC/MS-MS analysis was used to generate the TRP metabolomic profiles of mouse plasma, and the KYN/TRP ratio in the mouse plasma was calculated. I, Flow cytometry analysis of GZMB+ in CD8 + T cells. J, Flow cytometry analysis of CD25 + and Foxp3 + Tregs in CD4 + T cells. K, Representative flow cytometry histograms and statistical analysis of cell surface PD-1 with the indicated treatments. L and M, Representative images of tumors ( L ) and growth curves ( M ) of indicated Renca tumors in BALB/c mice ( n = 5 per group). N, Tumor weight measured after surgical dissection ( n = 5 per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The immortalized renal epithelial cell line (HK-2, RRID:CVCL_YE28), the human RCC cell lines [786-O (RRID: CVCL_1051), 769-P (RRID: CVCL_1050), A-498 (RRID:CVCL_1056), ACHN (RRID: CVCL_1067), and Caki-1 (RRID:CVCL_0234)] and the mouse RCC cell line (Renca, RRID:CVCL_2174) were purchased from ATCC.

Techniques: Western Blot, Control, Protein-Protein interactions, Expressing, Knockdown, Over Expression, Cell Culture, Enzyme-linked Immunosorbent Assay, Injection, Tandem Mass Spectroscopy, Clinical Proteomics, Flow Cytometry, Dissection

Journal: Cell metabolism

Article Title: Metabolic Maturation during Muscle Stem Cell Differentiation Is Achieved by miR-1/133a-Mediated Inhibition of the Dlk1-Dio3 Mega Gene Cluster.

doi: 10.1016/j.cmet.2018.02.022

Figure Lengend Snippet: Figure 3. Upregulation of Dlk1-Dio3 Cluster miRNAs Is Linked to Downregulation of Multiple Mitochondrial Genes (A–C) GSEA of miR-1/133a dKO and control TA muscles. ES, enrichment score. (D) Microarray-based transcriptional analysis of Dlk1-Dio3 cluster miRNAs and their potential targets (columns 1 and 2) in miR-1/133a dKO versus control TA muscles and RT-qPCR validation (column 3; n = 7/3 for miRNA assays, n = 3/3 [males] for mRNA assays, Mann-Whitney U test, one-tailed, p*%0.05, **p < 0.01). RT-qPCR analysis of mitochondrial target genes after transfection of Dlk1-Dio3 cluster miRNAs miR-382, miR-411, miR-541, and miR-543 into differentiating C2C12 myoblasts (columns 5–8) (n = 4/4 independent transfections; Mann-Whitney U test, one-tailed, p*<0.05). See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Real-Time PCR-Assay SLC25a23 Thermo Cat# Mm01232022_m1 Real-Time PCR-Assay Gapdh Thermo Cat# Mm99999915_g1; PL, VIC-labeled Deposited Data Satellite cells microarrays This paper Arrayexpress: E-MTAB-5892 M. tibialis anterior microarrays This paper Arrayexpress: E-MTAB-5936 Satellite cell RNA seq data This paper Geo: GSE108040 miRNA seq multiple cells and tissues Kuchen et al., 2010 Geo: GSE21630 Experimental Models: Cell Lines C2C12 cells ATCC CRL-1772 Sol8 cells ATCC CRL-2174 Mouse embryonic fibroblasts this paper N/A Experimental Models: Organisms/Strains Mouse: miR-1-1/miR-133a-2 KO Wystub et al., 2013 N/A Mouse: miR-1-2/miR-133a-1fl/fl this paper N/A Mouse: Pax7-Cre Keller et al., 2004 Mouse: Mef2A-tg this paper N/A Oligonucleotides See Table S2 Software and Algorithms GESA Subramanian et al., 2005 http://software.broadinstitute.org/gsea GraphPad Prism 6 Graph Pad Software, Inc N/A MaxQuant 1.5.3.30 Cox and Mann, 2008 http://www.biochem.mpg.de/5111795/maxquant Perseus 1.5.2.6 Cox and Mann, 2008 http://www.biochem.mpg.de/5111810/perseus

Techniques: Control, Muscles, Microarray, Quantitative RT-PCR, Biomarker Discovery, MANN-WHITNEY, One-tailed Test, Transfection