Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: TCIRG1 expression in different tumors and their cancerous tissues. ( A ) TCIRG1 expression levels in TCGA tumor tissues and TCGA and GTEX normal tissues. ( B ) Comparison of TCIRG1 expression between TCGA tumor data and the corresponding TCGA normal data. ( C ) RNA expression levels of TCIRG1 in tumor and normal tissues in renal clear cell carcinoma (KIRC) samples from the UALCAN database. GEO database series, including TCIRG1 expression levels in GSE66272 ( D ) and GSE53757 ( E ), and GSE15641 ( F – H ) immunohistochemistry staining statistics results for normal kidney tissues and KIRC tissues. Scale bar: 50 μm. (* p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no statistical significance).

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques: Expressing, Comparison, RNA Expression, Immunohistochemistry, Staining

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: Correlation between the expression of TCIRG1 and tumor prognosis ( A , C , E ). Kaplan–Meier curves demonstrating OS, PFI, and DSS of patients in the high- and low-TCIRG1 expression groups in KIRC samples. Forest plots demonstrate the prognostic HR of TCIRG1 in different cancer subgroups in OS ( B ), PFI ( D ), and DSS ( F ).

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques: Expressing

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: Correlation between high TCIRG1 expression and clinical traits. ( A ) Grade, ( B ) Stage, ( C ) T, ( D ) M, and ( E ) N correlation with TCIRG1 expression. ( F ) Heat map of correlation between clinical traits and TCIRG1 expression. ( G ) ROC curve to predict the diagnostic value of elevated TCIRG1. Nomogram for KIRC samples ( H ) and the calibration curve of the nomogram ( I ) for predicting OS at 1, 3, and 5 years. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques: Expressing, Diagnostic Assay

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: Analysis of OS using the Cox proportional hazards regression model.

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques:

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: Molecular characterization of high- and low-TCIRG1 expression subgroups. ( A , B ) Distribution of the ten most commonly mutated genes in the high-TCIRG1 TCGA-KIRC subgroup. The top bar shows the TMB of each patient, and the right bar shows the different types of mutations. Differences in TCIRG1 expression between ( C ) wild-type and PBRM1 mutant subgroups and ( D ) BAP1 mutant subgroups. ( E ) Correlation analysis of TCIRG1 expression with TMB. ( F ) Relationship between TCIRG1 expression and DNA methylation. ( G ) Relationship between DNA methylation levels and OS. ( H,I ) Correlation analysis of TCIRG1 expression with cancer stemness DNAss and RNAss. ( J ) Correlation analysis of RNAss levels with OS.

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques: Expressing, Mutagenesis, DNA Methylation Assay

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: Enrichment analysis of co-expressed genes of TCIRG1 in TCGA-KIRC. ( A ) Volcano maps showing the co-expression of TCIRG1 genes (FDR < 0.01, COR > 0.4). ( B ) The top fifty genes with the strongest correlation are shown in the heat map. ( C ) GO pathway analysis of TCIRG1 co-expressed genes in KIRC. ( D ) KEGG pathway analysis of TCIRG1 co-expressed gene in KIRC.

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques: Expressing

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: Correlation between TCIRG1 expression and tumor microenvironment score in KIRC. ( A ) BGN expression is associated with estimate score, Immunescore, and Stromalscore in renal cancer based on Pearson correlation analysis. ( B , C ) Relationship between OS of KIRC and Estimatedscore and Immunescore.(*** p < 0.001).

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques: Expressing

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: TIIC profile and correlation analysis in kidney cancer samples. Pie charts showing the estimated proportions of different types of TIICs in renal tumor samples predicted by ( A ) quanTIseq and ( B ) CIBERSOR. Pearson correlation matrix of the proportions of different TIICs in the microenvironment of renal cancer quantified by ( C ) quanTIseq and ( D ) CIBERSORT. The size of each bubble and the shading of each small colored box represents the corresponding correlation value between two cells. ( E , F ) Lollipop plots showing the correlation between TCIRG1 expression and different TIIC ratios analyzed by quanTIseq and CIBERSORT.

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques: Expressing

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: TCIRG1 and immune cell biomarkers in RCC were correlated using GEPIA2 and Timer2.0.

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques:

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: Division of subgroups according to TCIRG1 expression levels to predict potential immunotherapeutic responses in kidney cancer. ( A ) Immune checkpoint-associated genes are expressed in high- and low-TCIRG1 subpopulations. The Wilcoxon rank-sum test was used as a statistical significance test. ( B ) Tumor Immune Dysfunction and Exclusion (TIDE) score. ( C ) TCIRG1 expression differences between GSE67501-responding and non-responding groups. ( D – M ) IC50 differences between high- and low-TCIRG1 expression groups. (* p < 0.05, ** p < 0.01. *** p < 0.001).

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques: Expressing

Journal: Cancers

Article Title: Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cancers14194583

Figure Lengend Snippet: ( A ) Expression of TCIRG1 in different kidney cancer cell lines. TCIRG1 promotes the migratory ability of RCC cells. ( B , C ) qPCR Western Blot and ( D ) Western blotting showed that the expression of TCIRG1 was silenced by siRNA in 769P and caki1, respectively. Scratch wound-healing ( E , F , I , J ) and ( G , H , K , L ) transwell migration healing assays demonstrated TCIRG1-regulated migration ability. The scale bar of (E and I) is 250 μm, and the scale bar of (G and K) is 100 μm (* p < 0.05, *** p < 0.001).

Article Snippet: Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase.

Techniques: Expressing, Western Blot, Migration

Journal: Cancers

Article Title: Proteome-Wide Analysis Reveals TFEB Targets for Establishment of a Prognostic Signature to Predict Clinical Outcomes of Colorectal Cancer.

doi: 10.3390/cancers15030744

Figure Lengend Snippet: Figure 1. Mutations in S138 and S142 caused almost complete nuclear retention of TFEB. (A) Schematic of the human TFEB protein domains. S138 and S142 are localized in the proxim- ity of a NES. (B) S138 and S142 of TFEB are evolutionarily conserved in the indicated species. Alignment of the sequences near TFEB S138 and S142 is shown. (C) Western blots of endogenous and exogenous TFEB in CRC cells transfected with wild-type TFEB-GFP or TFEB-GFP mutant (S142A/S138A). Uncropped immunoblots are provided in the Figure S1. (D,E) Localization of TFEBWT and TFEBS142A/S138A in the cytosol and in the nucleus was detected by confocal microscopy. Representative images (D) and statistical results (E) are shown. Torin1 (250 nm), a positive control. Cell outlines in white dotted lines. DAPI was used to label the nucleus. Scale bars, 5 µm. Mean ± SD, n = 10 cells per condition, unpaired t-test. (F) Cell viability of indicated cells were analyzed using a CCK8 assay. Mean ± SEM, n = 3, unpaired t-test. ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies (against TFEB, GFP, LAMP2, NDRG1, SMPD1, and actin) and the HRP-conjugated secondary antibodies (goat anti-rabbit antibody) were supplied by Proteintech (Wuhan, China).

Techniques: Western Blot, Transfection, Mutagenesis, Confocal Microscopy, Positive Control, CCK-8 Assay