Journal: Cancer research

Article Title: Inhibition of Cholinergic Signaling Causes Apoptosis in Human Bronchioalveolar Carcinoma

doi: 10.1158/0008-5472.CAN-12-3190

Figure Lengend Snippet: Human BACs and normal HPAEpiCs express cholinergic proteins. A, ELISA assays show that human BAC cell lines and HPAEpiCs express multiple nAChR subunits. The assay was completed in duplicate, and the whole experiment was carried out 2 independent times. Results indicated by a different letter are significantly different (P < 0.05). B, HPAEpiCs and human BACs express AChE, ChAT, CHT1, and VAChT. H520 human SCC-L cells were used as the positive control for the experiments outlined in A and B. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control, and the results were quantitated by densitometric analysis. The experiment was repeated twice, and the representative data are shown. C and D, immunohistochemistry of the human BAC microarray showed that BAC tumors (isolated from patients) express ChAT and VAChT in the cytoplasm, adjacent to the hematoxylin-counterstained dark nuclei in the tissue samples. A tumor microarray containing 81 cores of human BACs was used for these experiments and 4 panels of representative photos are shown. Scale bar, 20 μm.

Article Snippet: Immunohistochemical staining of VAChT, ChAT in human BAC tissue microarray and normal lung TMA Human BAC tissue microarray (TMA) slides (Abnova) were deparaffinized and rehydrated as described previously ( 19 , 20 ).

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control, Control, Immunohistochemistry, Microarray, Isolation

Journal: Oncogenesis

Article Title: MicroRNA-18a is elevated in prostate cancer and promotes tumorigenesis through suppressing STK4 in vitro and in vivo

doi: 10.1038/oncsis.2014.12

Figure Lengend Snippet: miR-18a is highly expressed in tumors. ( a ) The expression level of miR-18a in prostate cancer tissue. The differential expression of miR-18a in prostate cancer tissues was compared with that in normal tissues. The level of miR-18a was significantly higher in cancer tissues ( P <0.01). ( b ) Left panel, ISH was used to detect the presence of miR-18a in non-tumor prostate and prostate cancer tissues. Representative ISH micrographs ( × 400) for miR-18a staining; right Panel, quantification according to ISH expression of miR-18a in prostate tumors as related to paired normal tissues. The scores are calculated as staining intensity × percentage of stained cells. ( c ) miR-18a relative expression level of non-tumor parts compared with tumor parts were analyzed using GEO data set. ( d ) The five NT paired tissues were used for examination of miR-18a expression. High expression of miR-18a tumor part specimens than that in non-tumor part tissues ( e ) The differential expression of miR-18a in prostate cancer cell lines was compared with that in prostate immortalized epithelium cell lines. The level of miR-18a was significantly higher in prostate cancer cell lines than in prostate immortalized epithelium cell lines.

Article Snippet: We used the prostate cancer tissue array obtained from BioMax Company (Rockville, MD, USA) to assay the correlation of miR-18a between TNM stage.

Techniques: Expressing, Quantitative Proteomics, Staining

Journal: Oncogenesis

Article Title: MicroRNA-18a is elevated in prostate cancer and promotes tumorigenesis through suppressing STK4 in vitro and in vivo

doi: 10.1038/oncsis.2014.12

Figure Lengend Snippet: STK4 is inversely correlated with miR-18a. ( a ) Venn diagram displaying miRNAs computationally predicted to target STK4 by PicTar (red), TargetScan (green), miRanda (blue) and Mirnatargets (black). Examination of different databases to find candidate downstream target genes. ( b ) Protein expression of STK4, SMAD2, CDC42, CTGF and α-tubulin in control miR or miR-18a precursor transfected prostate cancer cells (PC-3, DU145). STK4 was suppressed by miR-18a transfection at the protein level in prostate cancer cell lines as shown by western blotting. ( c ) The expression of STK4 mRNA (upper panel) and protein (bottom panel) in six prostate cell lines was analyzed by RT-PCR and western blotting, respectively. The number below the GAPDH image was the density ratio of STK4/GAPDH (NIH-Image J) for mRNA (upper panel) and protein (bottom panel), respectively. The STK4 mRNA level was the same in 60 cell lines. The STK4 protein level is downregulated in four prostate cancer cell lines. ( d ) Western blotting (upper panel) and IHC (middle panel) analysis revealed STK4 protein level was downregulated in prostate tumor parts compared with adjacent non-tumor tissues. Quantification of STK4 expression in paired prostate adenocarcinoma and corresponding normal tissues (bottom panel) ( n =10, P <0.001). Western blotting data showed that the STK4 protein is highly expressed in non-tumor compared with tumor tissues. IHC showed that STK4 staining is lost in the tumor tissues. ( e ) STK4 mRNA expression levels are not different between normal and tumor of prostate cancer in GEO data set validation. ( f ) ISH and IHC were used to detect the presence of miR-18a and STK4 in prostate cancer tissues. These sections of ISH and for IHC are consecutive sections. Quantitative IHC and ISH scores show that STK4 and miR-18a expression are inversely correlated. ( g ) miR-18a is highly expressed with poor tumor stage.

Article Snippet: We used the prostate cancer tissue array obtained from BioMax Company (Rockville, MD, USA) to assay the correlation of miR-18a between TNM stage.

Techniques: Expressing, Control, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Biomarker Discovery

Journal: Oncogenesis

Article Title: MicroRNA-18a is elevated in prostate cancer and promotes tumorigenesis through suppressing STK4 in vitro and in vivo

doi: 10.1038/oncsis.2014.12

Figure Lengend Snippet: miR-18a increased cell survival by suppressing STK4-mediated AKT kinase activity to inhibit apoptosis. ( a ) MTT assay indicating that the overexpression of STK4 and antagomiR-18a induces cell death in 22Rv-1 cells. ( b ) Western blotting of miRNA-transfected cells to detect the PARP cleavage level. ( c ) AKT phosphorylation in prostate cancer cells was suppressed by ectopic STK4 expression. Our observation in LNcap and 22Rv-1 prostate cancer cells transfected with STK4 suppressed AKT phosphorylation activity and increased PARP cleavage protein. ( d ) Representative images show that the expression levels of STK4 are inversely related to the phosphorylation levels of AKT in prostate cancer xenograft tumor tissues. ( e ) Diagram indicating how miR-18a regulates STK4 to act as an oncomiR in prostate cancer.

Article Snippet: We used the prostate cancer tissue array obtained from BioMax Company (Rockville, MD, USA) to assay the correlation of miR-18a between TNM stage.

Techniques: Activity Assay, MTT Assay, Over Expression, Western Blot, Transfection, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: HCP5 prevents ubiquitination-mediated UTP3 degradation to inhibit apoptosis by activating c-Myc transcriptional activity

doi: 10.1101/2022.06.13.495862

Figure Lengend Snippet: A Volcano plots showing upregulated and downregulated ncRNAs in KYSE450/DDP and YES2/DDP cells compared to their parental cells. B RT–qPCR detection of HCP5 expression in the parental and DDP-resistant cell models. The data are presented as the means ± s.d.; two-tailed t test, *** P < 0.001; n = 3. C RT–qPCR detection of HCP5 expression in KYSE30 and KYSE450 cells treated with DDP for 0, 6, 12, and 24 h. The data are presented as the means ± s.d.; two-tailed t test, *** P < 0.001, ** P < 0.01, * P < 0.05; n = 3. D Overall survival of ESCC patients with low and high HCP5 expression levels (divided by the mean expression value). Kaplan–Meier survival plots are shown. E Statistical analysis of HCP5 expression in 168 paired ESCC tissues and adjacent normal tissues. Box plot representation: from top to bottom—maximum, 75th percentile; median, 25th percentile; and minimum values; two-tailed t test. F Statistical analysis of HCP5 expression in 53 paired ESCC tissues and adjacent normal tissues in the GEO database. Box plot representation: from top to bottom—maximum, 75th percentile; median, 25th percentile; and minimum values; two-tailed t test. G-J Tumor volumes and representative images (G and I) and tumor weights (H and J) of the xenografts derived from the indicated cells. The data are presented as the means ± s.e.m.; two-tailed t test, *** P < 0.001, ** P < 0.01; n = 7. K-M Tumor weights in the three PDX models from ESCC patients treated with AAV-shCtrl or AAV-shHCP5. The data are presented as the means ± s.e.m.; two-tailed t test, *** P < 0.001; n = 8.

Article Snippet: For ISH, human ESCC tissue microarray slides were purchased from Servicebio (#G6040, Wuhan, Hubei, China).

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: HCP5 prevents ubiquitination-mediated UTP3 degradation to inhibit apoptosis by activating c-Myc transcriptional activity

doi: 10.1101/2022.06.13.495862

Figure Lengend Snippet: A FISH detection of HCP5 localization in KYSE150 and KYSE510 cells. Nuclei were stained with Hoechst 33342. Scale bar, 30 μm. B List of HCP5-interacting proteins identified using MS with KYSE150 and KYSE510 cells. C Western blot analyses of UTP3 in precipitates from RNA pull-down assays performed using antisense or sense sequences of HCP5. D RT–qPCR measurement of the HCP5 level in precipitates from RIP assays performed using IgG or anti-UTP3 antibodies. The data are presented as the means ± s.d.; two-tailed t test, *** P < 0.001; n = 3. E Correlation analysis of HCP5 and UTP3 protein expression in ESCC cell lines. Spearman correlation coefficients are shown. F Western blot analyses of UTP3 and MCL1 expression in control and HCP5-silenced KYSE150 and KYSE510 cells treated with DMSO or MG132 for 8 h. G Western blot analyses of UTP3 expression in control and HCP5-silenced KYSE150 and KYSE510 cells subjected to a CHX pulse-chase assay. H Quantitative analyses of UTP3 expression in control and HCP5-silenced KYSE150 and KYSE510 cells subjected to the CHX pulse-chase assay. Quantification of UTP3 expression relative to β -actin expression is shown. The data are reported as the means ± s.e.m.; n = 3. I, J Western blot detection to assess UTP3 ubiquitination in control and HCP5-silenced KYSE150 (I) and KYSE510 cells (J).

Article Snippet: For ISH, human ESCC tissue microarray slides were purchased from Servicebio (#G6040, Wuhan, Hubei, China).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Two Tailed Test, Expressing, Pulse Chase

Journal: bioRxiv

Article Title: HCP5 prevents ubiquitination-mediated UTP3 degradation to inhibit apoptosis by activating c-Myc transcriptional activity

doi: 10.1101/2022.06.13.495862

Figure Lengend Snippet: A, B GSEA results based on RNA-seq data comparing HCP5-silenced to control KYSE150 (A) and KYSE510 (B) cells. C Prediction of the sequence logo of the MYC-binding site based on the JASPAR database. D Predicted binding sequence of MYC in the VAMP3 promoter region, as determined from the JASPAR database. E Schematic showing MYC binding sites at the VAMP3 promoter region and the unique peaks identified by ATAC-seq. F, G ChIP analyses of the indicated cells using antibodies against c-Myc to identify the indicated gene promoter segments. The data are presented as the means ± s.d.; two-tailed t test, *** P < 0.001, ** P < 0.01; n = 3. H RT–qPCR detection of the expression of the indicated genes in samples from ESCC patients with chemoresistance or chemosensitivity. The data are presented as the means ± s.e.m.; two-tailed t test; n = 17 for chemoresistant patients and n = 17 for chemosensitive patients. I Correlation analyses between the indicated gene expressions in ESCC samples. Spearman correlation coefficients are shown.

Article Snippet: For ISH, human ESCC tissue microarray slides were purchased from Servicebio (#G6040, Wuhan, Hubei, China).

Techniques: RNA Sequencing Assay, Sequencing, Binding Assay, Two Tailed Test, Quantitative RT-PCR, Expressing

Journal: Frontiers in Oncology

Article Title: PGC1β Regulates Breast Tumor Growth and Metastasis by SREBP1-Mediated HKDC1 Expression

doi: 10.3389/fonc.2019.00290

Figure Lengend Snippet: HKDC1 expression involves with tumor growth and metastasis in vivo . (A,B) Tissue Microarray slides (#CHTN BrCaProg1) were obtained from CHTN (Cooperative Human Tissue Network), and the immunohistochemistry was performed using a primary rabbit antibody for either PGC1β or HKDC1 and a second anti-rabbit- FITC. Sixty cells from normal breast tissue (normal), primary tumor (tumor), and metastatic (metastasis) tissues were quantitated by Image J. (A) Protein quantitation, n = 3. *, P < 0.05, vs. Normal group; ¶, P < 0.05 vs. Tumor group. (B) Representative pictures for (A) . (C–H) The nude mice were injected with treated MCF7 cells through the tail vein for in vivo xenograft tumor development study, and the treated mice were sacrificed for further analysis. (C) The tumor tissues from the lung were isolated for mRNA analysis by qPCR, n = 4. *, P < 0.05, vs. CTL group. (D) Superoxide anion release from tumor tissues, n = 5, *, P < 0.05, vs. CTL group; #, P < 0.05, vs. shPGC1β group. (E–G) Mice were killed upon 20% weight loss, and the lungs were harvested for terminal analysis. The metastatic tumor nodules from the lungs were counted, and then the formalin-fixed, paraffin-embedded tumor tissue of the lung was sectioned to 4 mm thickness, and the histopathological analyses were performed with H&E staining. Images were taken using a Carl Zeiss MIRAX MIDI slide scanner, and the lung tumor spots were analyzed using a 3DHISTECH Pannoramic Viewer. (E) Tumor colony formation in lung, n = 9. *, P < 0.05, vs. CTL group; ¶, P < 0.05, vs. ↑PGC1β group; #, P < 0.05, vs. shPGC1β group. (F) Quantitated lung tumor spots, n = 5. *, P < 0.05, vs. CTL group; ¶, P < 0.05, vs. ↑PGC1β group; #, P < 0.05, vs. shPGC1β group. (G) Representative picture by H&E staining. (H) Kaplan-Meier analysis comparing survival of mice between each treatment group, P -value represents log-rank Mantel-Cox test result, n = 9. Results are expressed as mean ± SEM.

Article Snippet: CHTN BrCaProg1 Microarray slides were obtained from CHTN, including normal, tumor, and metastatic breast cancer tissues.

Techniques: Expressing, In Vivo, Microarray, Immunohistochemistry, Protein Quantitation, Injection, Isolation, Formalin-fixed Paraffin-Embedded, Staining