Journal: Nucleic Acids Research

Article Title: A transcriptomic and proteomic map of primary human cell types

doi: 10.1093/nar/gkaf1498

Figure Lengend Snippet: Cell type specific genes demonstrated at mRNA level. (A) Heat map of genes with cell type specific expression. (B) Heat map of selected genes with endothelial or epithelial cell type specific expression. (C) Immunohistochemistry staining of protein C1orf116 across human tissues showing its epithelial cell type specific expression. The panel shows a magnified view (20×) from tissue microarrays.

Article Snippet: The Human Protein Atlas (HPA) project provided a tissue-based map of the human proteome through transcriptomics and tissue microarray-based immunohistochemistry analysis [ , ].

Techniques: Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) The Cancer Genome Atlas (TCGA) database analyses demonstrate RBM3 mRNA levels are significantly higher in colon cancer (T) (n=286) than in normal colon (N) (n=41) (p<0.001). (B) RBM3 mRNA expression from a cDNA array of colon tumor (T) samples (n=24) and matched adjacent normal colon tissue (N) (n=24) normalized to β-actin, shows a significant increase of RBM3 expression in tumor tissue (p=0.023). (C) Immunohistochemistry (IHC) of a colon cancer tumor microarray shows that RBM3 is upregulated in colon adenocarcinoma along with lymph node metastasis and liver metastasis as compared to the normal colon, lymph node, and liver. (D) Composite score of colon cancer TMA shows significantly higher expression of RBM3 in tumor (n=28) (p=0.002) and metastasis (n=30) (p=0.036) as compared to normal tissue. RBM3 expression is also increased in the different stages of colon cancer (Stage I (n = 3), Stage II (n = 12) (p=0.015), Stage III (n= 11) (p=0.07), Stage IV (n = 2) and metastasis (n=30) (p=0.036)) as compared to normal colon, liver and lymph node (n=3 each). (E) Western blot analysis of RBM3 protein expression in established colon cancer cell lines HCT116, DLD1, SW480, SW620, HT29, RKO and LST17T as compared to normal colon epithelial cells (FHC cell line). Data in are represented as ± SEM. Also, see Supplementary figure 1

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Expressing, Immunohistochemistry, Microarray, Western Blot

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) Volcano plots for RNA-sequencing (RNA-seq) showing differentially expressed lncRNA in both HCT116 and DLD1 RBM3 overexpressing (RBM3 O/E) cell lines compared to empty vector (EV) cell lines. (B) Venn diagram depicting the number of co-expressed and uniquely expressed lncRNA found in RNA-seq. (C) The volcano plots for RNA-immunoprecipitation coupled sequencing (RNA-IP seq) showing differentially expressed lncRNA in both HCT116 and DLD1, RBM3 overexpressing cell lines compared to empty vector cell lines. (D) Venn diagram depicting the number of co-expressed and uniquely expressed lncRNA found in RNA-IP seq. (E-F) Plot from REVIGO software showing the Gene Ontology (GO) terms enriched in lncRNA (RNA-seq) for HCT116 RBM3 (E). The graph has been modified to highlight the GO terms and color key. The box plot drawn highlighting enriched GO terms(F). (G) RT-PCR validation of lncRNA identified through RNA-seq in colon cancer cell lines compared to normal FHC cells. (H) RT-PCR validation of lncRNA identified through RNA-seq, in the RBM3 overexpressing (RKO, HCT116, and DLD1) cells, show increased expression of lnc-HOTAIR, lnc-TUG1, lnc-Flii- 1, lnc-LSAMP-3. (I) Western blot analysis for the expression demonstrating levels of RBM3 in the distal colon in representative wild type littermates and RBM3 transgenic mice (RBM3 O/E). (J) RT-PCR analysis demonstrating increased expression of lncRNA lnc-Flii-1 and lnc-LSAMP- 3 compared to GAPDH in representative wild type (WT) littermates and RBM3 overexpressing transgenic mice (RBM3 O/E). Data in are represented as ± SEM. Also, see Supplementary figure 2.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: RNA Sequencing, Plasmid Preparation, RNA Immunoprecipitation, Sequencing, Software, Modification, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Western Blot, Transgenic Assay

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A-D) Quantitative PCR shows increased mRNA levels of VEGFA, ZEB1, TWIST, and SNAI2 in RBM3 overexpressing cells compared to empty vector cell lines. (E) Plot for tube length of the endothelial tubular network formed by HUVEC cell in (G). Increased tube formation in HUVEC cells treated by condition media from HCT116 (p=0.038) and DLD1 (p=0.004) RBM3 overexpressing compared to empty vector cells (F) Plot showing increase in migration of HCT116 (p=0.001), DLD1 (p=0.02) and RKO (p=0.04) RBM3 overexpressing cells compared to empty vector performed by wound healing assay. (G) Plot showing increase in migration of HCT116 (p=0.006), DLD1 (p=0.002) and RKO (p=0.006) RBM3 overexpressing cells compared to empty vector performed by transwell migration assay. (H) Plot showing increase in invasion of HCT116 (p<0.001), DLD1 (p<0.01) and RKO (p<0.01) RBM3 overexpressing cells compared to empty vector performed by transwell invasion assay. Data in are represented as ± SEM. Also, see Supplementary figure 3.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Migration, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A-D) The plot of tumor weights (A, C) and tumor volumes (B, D) from HCT116 and DLD1 empty vector and RBM3 overexpressing xenografts. The tumor volume and weight were significantly increased in RBM3 overexpression compared to control (p<0.05) (E) RT-PCR validation of lncRNA in the tumor xenograft tissues, in both HCT116 and DLD1 xenografts. RBM3 overexpressing xenografts show an increase in lnc-Flii-1 and lnc-LSAMP-3 expression compared to empty vector. (F) RT-PCR validation for increased expression of TWIST1, SNA2 and VEGFA in HCT116 and DLD1 empty vector and RBM3 overexpressing xenograft tumors tissues. (G) Western blot analysis of tumors from HCT116 and DLD1 xenograft tissues for VEGF expression. (H) Increassed RBM3 and PCNA levels in HCT116 and DLD1 RBM3 overexpressing xenograft tissues as compared to empty vector as seen by immunohistochemistry analysis. (I) Increase in percentage of PCNA positive nuclei in RBM3 overexpressing HCT116 (p=0.022) and DLD1 (p=0.025) xenograft tissues immunohistochemistry. The data for are presented as the means ± SEM, (n=10). Also, see Supplementary figure S5.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Plasmid Preparation, Over Expression, Control, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Western Blot, Immunohistochemistry

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) Protein expression of RBM3 decreases in shRNA knockdown clones (2,3,5) compared to scramble (scr1,scr2) in the HCT116 cell line by Western blot analysis. (B) RT-PCR validation for the decreased expression of lnc-LSAMP-3 and lnc-Flii-1 in HCT116 RBM3 shRNA knocked down clone compared to scramble. (C) Plot for decrease in percentage proliferation of the RBM3 shRNA knockdown clones over empty vector for sh2 (proliferation= 55.06%, p=0.04), sh3 (proliferation= 53.8%, p=0.0023), sh5 (proliferation= 47.45%, p=0.0001). (D) The plot showing decrease in percent migration in HCT116 shRNA knocked down clones (sh2 p=0.044 and sh5 p=0.048) compared to scramble performed by Transwell migration. (E) The plot showing decrease in percent invasion in HCT116 shRNA knocked down clones (sh2 p=<0.001 and sh5 p=0.001) compared to scramble performed by Transwell invasion assay. (F and G) The plot of tumor weights (F) and tumor volumes (G) from HCT116 scramble (scr2) and RBM3 shRNA knockdown (shRNA-2) xenografts. Both tumor volume and weight were significantly reduced in RBM3 knockdown compared to scramble control (p<0.05). (H) Kaplan-Meier analysis showing increased percent survival after the AOM/DSS induced carcinogenesis in cre inducible RBM3 knockout (KO) compared to wild type (WT) mice. (I) Plot showing decreased intestinal permeability in RBM3 knockout mice as compared to the wild type littermates (p=0.04). (J) The plot showing decreased number of tumors in the distal colon observed after the AOM/DSS induced carcinogenesis in cre inducible RBM3 knockout compared to wild type mice (p=0.007). (K) Representative image of H and E staining of the distal colon with tumors after the AOM/DSS induced carcinogenesis in RBM3 knockout and wild type mice. (L) Decrease in expression of lnc-LSAMP-3 and lnc-Flii-1 seen by PCR analysis in RBM3 knockout mice as compared to the wild type mice. The data for are presented as the means ± SEM, (mice n=10). Also, see Supplementary figure 6.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Expressing, shRNA, Knockdown, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Plasmid Preparation, Migration, Transwell Invasion Assay, Control, Knock-Out, Permeability, Staining

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A and B) RT-PCR validation of lnc-LSAMP-3 and lnc-Flii-1 knocked down in HCT116 (A) and DLD1 (B) RBM3 O/E and empty vector cells. (C and D) RT-PCR validation of VEGFA, ZEB1 and TWIST in HCT116 (C) and DLD1 (D) RBM3 O/E and empty vector cells, after lncRNA (lnc-Flii-1 and lnc-LSAMP-3) knockdown. (E) Plot showing decreased percent migration of HCT116 empty vector and RBM3 overexpressing cells having knockdown of lncRNA lnc-LSAMP-3 (Empty vector p=0.017, RBM3 O/E p=0.031) and lnc-Flii-1(Empty vector p=ns, RBM3 O/E p=0.011) performed by scratch plate assay. (F) Plot showing decreased tube length of the endothelial tubular network formed by HUVEC cell treated by condition media from knockdown of lncRNA lnc-LSAMP-3 and lnc-Flii-1(Empty vector p=0.012, RBM3 O/E p=0.022) in HCT116 cells. (G-J)The plot showing decrease in tumor weight (G, I) and tumor volumes (H, J) from HCT116 and DLD1 xenografts treated with intratumoral injection of si+ LNA gapmer for lnc-Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control (p< 0.05). (K and L) RT-PCR validation for the decreased expression of lncRNA (lnc-LSAMP-3 and lnc- Flii-1) in the tumor xenograft of HCT116 (K) and DLD1(L) treated with intratumoral injection of si+ LNA gapmer for lnc-Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control. (M and N) Western blot showing decreased protein expression of SNAI2 and VEGF in HCT116 (M) and DLD1 (N) tumor xenograft treated with intratumoral injection of si+ LNA gapmer for lnc- Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control. The data for are presented as the means ± SEM. Also, see figure Supplementary figure

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Plasmid Preparation, Knockdown, Migration, Injection, Control, Expressing, Western Blot

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited the proliferation and induced the apoptosis of PCa cells. The IC50 of mannose in ( a ) DU145 and ( b ) PC3 cells was determined using a CCK-8 assay. ( c ) Intracellular mannose concentration in PCa cells. Cell proliferation of ( d ) DU145 and ( e ) PC3 was assessed using growth curves, respectively. ( f ) Colony formation assays were performed and ( g ) colony numbers were counted in PCa cells. ( h ) Flow cytometric analysis was used to assess ( i ) the apoptosis rate of PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; IC50: the half-maximal inhibitory concentration; CCK-8: Cell Counting Kit-8; AAD: Aminoactinomycin D; APC: Allophycocyanin.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: CCK-8 Assay, Concentration Assay, Cell Culture, Cell Counting

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited tumor growth in a PCa xenograft model without affecting mice health. ( a ) Subcutaneous tumors from the xenograft model with DU145 cells. ( b ) Tumor growth was monitored for 30 days after mannose treatment. ( c ) The volume of tumor growth. ( d ) The weight of the tumors. ( e ) Intratumoral mannose concentration and ( f ) ATP content in subcutaneous tumors. The weights of ( g ) mice and ( h ) major metabolic organs. ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Concentration Assay, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted mitochondrial function, led to ROS overproduction, and activated Bax/Bak in PCa cells. ( a ) JC-1 staining and ( b ) rhodamine 123 staining were used to assess the MMP in DU145 and PC3 cells. ( c ) The ATP content in cells. ( d ) Mitochondrial ROS and ( e ) cellular ROS levels in cells. ( f and g ) The protein expression of Bax and Bak in cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine; Bax: BCL2-associated X; Bak: BCL2-antagonist/killer 1; ROS: reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine triphosphate; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Expressing, Cell Culture, Membrane

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted the balance of mitochondrial dynamics in PCa cells. ( a ) Mitochondria stained with MitoTracker Red were observed by confocal microscopy. ( b ) Mitochondrial structure under a transmission electron microscopy, and the mitochondrial cross-sectional area was quantified. ( c ) The protein expression of FIS1 in cells. Upregulated ( d ) FIS1 and ( e ) increased ATP content in PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. ATP: adenosine triphosphate; FIS1: fission, mitochondrial 1; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Expressing, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Downregulation of MPI expression enhances the anticancer effect of mannose. The expression of MPI in human prostate cancer tissues and its prognostic value. ( a and b ) The expression of MPI was silenced by siRNA-MPI and verified by western blotting. siRNA-MPI with different base sequences including si-1, si-2 and si-3 were used for downregulating the MPI expression in PCa cells. According to the degree of down-regulation of MPI protein, si-3 and si-2 with the best interference effect were applied to DU145 and PC3 cells, respectively. ( c ) Intracellular mannose concentration and ( d ) ATP content in cells. ( e ) Growth curves and ( f ) colony formation assays of cells. ( g ) The IHC scores for MPI expression in PCa tissues. ( h ) Kaplan–Meier curves of BCR-free survival and ( i ) overall survival for the low and high MPI expression groups of patients in the TCGA-PRAD dataset. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. MPI: mannose phosphate isomerase; si: siRNA, small interfering RNA; IHC: immunohistochemistry; PCa: prostate cancer; BCR: biochemical recurrence; TCGA-PRAD: The Cancer Genome Atlas-Prostate Adenocarcinoma; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TMA: tissue microarray; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Expressing, Western Blot, Concentration Assay, Cell Culture, Small Interfering RNA, Immunohistochemistry, Microarray

Journal: Nature communications

Article Title: RNF208, an estrogen-inducible E3 ligase, targets soluble Vimentin to suppress metastasis in triple-negative breast cancers.

doi: 10.1038/s41467-019-13852-5

Figure Lengend Snippet: Fig. 1 RNF208 is significantly underexpressed in highly metastatic breast cancer cells. a Volcano plot of RNF208 expression in luminal subtype (MCF-7, T47D, and ZR-75B) and TNBC (MDA-MB-231 and Hs578T) cells using RNA sequencing (GSE100878). b RT-PCR and immunoblot analysis of RNF208 expression in breast cancer cells. c Scatter plots of RNF208 expression in 52 breast cancer cells from public microarray datasets (GSE41313) (Luminal N = 29 and TNBC N = 23 cells). d, e Heat map and box plots showing the expression levels of RNF208, ESR1, and ERBB2 across the breast cancer subtypes using Genomic Data Commons (GDC). (Luminal A N = 335, Luminal B N = 88, HER2 N = 23 and TNBC N = 55 patients). f Comparison of RNF208 expression in breast cancer subtypes using published microarray datasets (GSE2034) (Luminal N = 154 and TNBC N = 55 patients). g Representative immunohistochemical (IHC) staining of RNF208 protein expression classified in luminal and basal breast cancer patient tissues. Original magnification ×100. Scale bar, 50 μm. h Real-time quantitative RT-PCR of RNF208 expression in luminal subtype tissues (N = 6) and TNBC tissues (N = 6); 18S rRNA was used as an internal control. i Kaplan−Meier analysis showing relapse-free survival depending on RNF208 expression levels from public meta-analysis data (N = 3951). P values were calculated using a log-rank test. All P values were calculated by unpaired two-tailed Student’s t tests (c, e, f, and h). These data represent the mean ± S.D. Source data for (c, f, h) is available in Source Data file. Unprocessed original scans of blots and gels in (b) are shown in Supplementary Fig. 13.

Article Snippet: The human breast cancer cell lines MCF-7, T47D, Hs578T, and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC); ZR-75B, cloned from the ZR-75-1 cell lines26, was obtained from the National Cancer Institute (NCI)/NIH.

Techniques: Expressing, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Microarray, Comparison, Immunohistochemical staining, Immunohistochemistry, Quantitative RT-PCR, Control, Two Tailed Test

Journal: Nature communications

Article Title: RNF208, an estrogen-inducible E3 ligase, targets soluble Vimentin to suppress metastasis in triple-negative breast cancers.

doi: 10.1038/s41467-019-13852-5

Figure Lengend Snippet: Fig. 2 RNF208 expression was transcriptionally activated by ERα in luminal breast cancer cells. a Comparison of RNF208 expression between ERα- positive and -negative breast cancer samples in published microarray datasets (GSE2034 and GSE5460). b RT-PCR (top) and immunoblot (bottom) analysis of ERα target genes (FOXOM1, GREB1, ESR1) and RNF208 expression in EtOH- or E2-treated ERα-positive breast cancer cells. 10 nM or 20 nM of E2 was treated in MCF-7 or T47D cell, respectively and E2 was treated for 24 h for RT-PCR and 48 h for immunoblot analysis. GAPDH and β-actin were used as internal controls. c Real-time quantitative RT-PCR (qRT-PCR) of RNF208 expression in ESR1-knockdown T47D cells upon E2 treatment. d Illustration of luciferase reporters including ERα-binding sites in the RNF208 promoter sequences. e T47D cells were transfected with various deletion constructs of the RNF208 promoter and then treated with or without E2 for 24 h. After E2 treatment, cells were assayed for luciferase activity. f ChIP analysis showing the recruitment of ERα to the human RNF208 promoter in E2-treated T47D cells. Precipitation was conducted with antinormal IgG or anti-ERα antibodies. g T47D cells were transfected with pGL3 control, RNF208 promoter, or its mutant (mtR4) plasmids containing mutated estrogen-responsive element site (CACC sequence replaced by GAAA) and then subjected to luciferase assays. h RT-PCR and immunoblot analysis showing RNF208 expression upon E2 treatment for 24 h (mRNA) or 48 h (protein), respectively, in control or ERE knockout MCF-7 cells. i Immunoblot analysis of ERα with 5-aza-dC treatment in TNBC cells. Hs578T and MDA-MB-231 cells were treated with 10 μM 5-aza-dC for 96 h and β-actin was used for normalization. j TNBC cells were transiently transfected with 20 nM of control siRNA or ESR1 siRNA and then treated with 10 μM 5-aza-dC for 96 h. Cell lysates were immunoblotted with the indicated antibodies. All P values were calculated by unpaired two-tailed Student’s t tests (e, g). These data represent the mean ± SD of three independent experiments. Source data for (c, e, g, h) are available in Source Data file. Unprocessed original scans of blots and gels in (b, f, h−j) are shown in Supplementary Fig. 13.

Article Snippet: The human breast cancer cell lines MCF-7, T47D, Hs578T, and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC); ZR-75B, cloned from the ZR-75-1 cell lines26, was obtained from the National Cancer Institute (NCI)/NIH.

Techniques: Expressing, Comparison, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Knockdown, Luciferase, Binding Assay, Transfection, Construct, Activity Assay, Control, Mutagenesis, Sequencing, Knock-Out, Two Tailed Test

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 2. Loss of ARG1 and AGMAT enhances liver tumor formation (A) Immunoblots of arginine-to-polyamine-converting enzymes (ARG1 and AGMAT) and polyamine metabolism enzymes (ODC, SRM, SMS, SAT1, PAOX, and SMOX) in Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control (same samples were used as in Figure 1E). n = 4 (Ctrl), n = 8 (L-dKO). (B) Total polyamine content in Ctrl liver and L-dKO tumor tissues. n = 6. (C) Relative 3H-putrescine uptake into Ctrl liver and L-dKO tumor tissues. n = 8. (D) Immunohistochemistry of Ctrl and L-dKO liver tissues stained for ARG1 or AGMAT. NT, adjacent non-tumor tissue; T, tumor. (E) Representative images of livers from L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. (F) Number of macroscopic tumors per liver of L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 9–10. (G) Arginine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 4–10. *p < 0.05, **p < 0.01. ***p < 0.001, ****p < 0.0001 by unpaired t test (B and C) and one-way ANOVA (F and G).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER L-Arginine-coupled agarose beads gbiosciences Cat#GENO786-1361 L-leucine-coupled agarose beads gbiosciences Cat#GENO786-1370 Critical commercial assays Total Polyamine Assay Kit BioVision Cat#K475 L-arginine ELISA kit MyBiosource Cat#MBS728648-96 Nano-Glo dual luciferase reporter assay kit Promega Cat#N1610 Deposited data HCC patients’ RNA sequencing data Ng et al., 202250 EGAS00001005073, EGAS00001005074 HCC patients’ proteome data Ng et al., 202250 PRIDE (PXD025705, PXD025836) Mouse RNA sequencing data Dimitrakopoulos et al., 202125 SRP156216 Mouse proteome data Dimitrakopoulos et al., 202125 https://github.com/cbgethz/netics/tree/ master/mouse_data Mouse and HCC patients’ metabolomics This paper MASSIVE: MSV000092406 RNA sequencing data of ARG1/AGMAT-expressing (and control) and RBM39-depleted (and control) SNU-449 cells This paper GEO: PRJNA940402 Mouse, HCC patients, and SNU-449 proteome data of pulldowns using Leu- and Arg-coupled agarose beads This paper MASSIVE: MSV000091516 Experimental models: Cell lines SNU-182 Diego Calvisi (University of Sassari, Italy) N/A SNU-449 Diego Calvisi (University of Sassari, Italy) N/A HLE Diego Calvisi (University of Sassari, Italy) N/A PLC Diego Calvisi (University of Sassari, Italy) N/A Hep40 Diego Calvisi (University of Sassari, Italy) N/A Hep3B Diego Calvisi (University of Sassari, Italy) N/A Huh6 Diego Calvisi (University of Sassari, Italy) N/A Huh7 Diego Calvisi (University of Sassari, Italy) N/A HepG2 Diego Calvisi (University of Sassari, Italy) N/A SNU-475 Gerhard Christofori (University of Basel, Switzerland) N/A SNU-423 Gerhard Christofori (University of Basel, Switzerland) N/A Huh1 Gerhard Christofori (University of Basel, Switzerland) N/A HEK293 ATCC CRL-1573 HEK239T ATCC CRL-3216 SNU-449 control This paper N/A SNU-449 ARG1 This paper N/A SNU-449 AGMAT This paper N/A SNU-449 ARG1/AGMAT This paper N/A SNU-449 ASS1-FLAG This paper N/A SNU-449 ARG1/AGMAT +ASS1 This paper N/A SNU-449 ARG1/AGMAT +3xHA-ASS1 This paper N/A Huh7 sgCtrl This paper N/A Huh7 sgARG1 This paper N/A Huh7 sgAGMAT This paper N/A SNU-449 ARG1/AGMAT +control (for ASNS) This paper N/A (Continued on next page) ll OPEN ACCESS Cell 186, 5068–5083.e1–e12, November 9, 2023 e2 Article

Techniques: Western Blot, Control, Immunohistochemistry, Staining, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 3. ARG1/AGMAT determine metabolic gene expression via arginine (A) Immunoblots of SNU-449 cells upon stable expression of ARG1 and/or AGMAT. Actin serves as loading control. (B) Representative clonogenic growth assay of control, ARG1-, and/or AGMAT-expressing SNU-449 cells grown in arginine-restricted medium. (C) Relative clonogenic growth of control, ARG1-, and/or AGMAT- expressing SNU-449 cells. N = 6. (D) Arginine content of control, ARG1-, and/or AGMAT-expressing SNU-449 cells. N = 4. (E) PCA analysis of RNA-seq data of control and ARG1/AGMAT-expressing SNU-449 cells. (F) Heatmap of a subset of differentially expressed metabolic genes in ARG1/AGMAT-expressing compared to control SNU-449 cells (log2 fold-change). (G) mRNA levels of ASNS, PSAT1, PSPH, GLSK, GLUT3, HK2, NNMT, and AOC3 in control and ARG1/AGMAT-expressing SNU-449 cells. N = 5–7. (H) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ARG1/AGMAT-expressing SNU-449 cells. Calnexin serves as loading control. (I) Immunoblots of ASNS, PSAT, PSPH, and NNMT of Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control. n = 4 (Ctrl), n = 8 (L-dKO). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (C and D) and unpaired t test (G).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER L-Arginine-coupled agarose beads gbiosciences Cat#GENO786-1361 L-leucine-coupled agarose beads gbiosciences Cat#GENO786-1370 Critical commercial assays Total Polyamine Assay Kit BioVision Cat#K475 L-arginine ELISA kit MyBiosource Cat#MBS728648-96 Nano-Glo dual luciferase reporter assay kit Promega Cat#N1610 Deposited data HCC patients’ RNA sequencing data Ng et al., 202250 EGAS00001005073, EGAS00001005074 HCC patients’ proteome data Ng et al., 202250 PRIDE (PXD025705, PXD025836) Mouse RNA sequencing data Dimitrakopoulos et al., 202125 SRP156216 Mouse proteome data Dimitrakopoulos et al., 202125 https://github.com/cbgethz/netics/tree/ master/mouse_data Mouse and HCC patients’ metabolomics This paper MASSIVE: MSV000092406 RNA sequencing data of ARG1/AGMAT-expressing (and control) and RBM39-depleted (and control) SNU-449 cells This paper GEO: PRJNA940402 Mouse, HCC patients, and SNU-449 proteome data of pulldowns using Leu- and Arg-coupled agarose beads This paper MASSIVE: MSV000091516 Experimental models: Cell lines SNU-182 Diego Calvisi (University of Sassari, Italy) N/A SNU-449 Diego Calvisi (University of Sassari, Italy) N/A HLE Diego Calvisi (University of Sassari, Italy) N/A PLC Diego Calvisi (University of Sassari, Italy) N/A Hep40 Diego Calvisi (University of Sassari, Italy) N/A Hep3B Diego Calvisi (University of Sassari, Italy) N/A Huh6 Diego Calvisi (University of Sassari, Italy) N/A Huh7 Diego Calvisi (University of Sassari, Italy) N/A HepG2 Diego Calvisi (University of Sassari, Italy) N/A SNU-475 Gerhard Christofori (University of Basel, Switzerland) N/A SNU-423 Gerhard Christofori (University of Basel, Switzerland) N/A Huh1 Gerhard Christofori (University of Basel, Switzerland) N/A HEK293 ATCC CRL-1573 HEK239T ATCC CRL-3216 SNU-449 control This paper N/A SNU-449 ARG1 This paper N/A SNU-449 AGMAT This paper N/A SNU-449 ARG1/AGMAT This paper N/A SNU-449 ASS1-FLAG This paper N/A SNU-449 ARG1/AGMAT +ASS1 This paper N/A SNU-449 ARG1/AGMAT +3xHA-ASS1 This paper N/A Huh7 sgCtrl This paper N/A Huh7 sgARG1 This paper N/A Huh7 sgAGMAT This paper N/A SNU-449 ARG1/AGMAT +control (for ASNS) This paper N/A (Continued on next page) ll OPEN ACCESS Cell 186, 5068–5083.e1–e12, November 9, 2023 e2 Article

Techniques: Gene Expression, Western Blot, Expressing, Control, Growth Assay, RNA Sequencing

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 4. ASNS promotes arginine uptake in liver cancer (A) Relative 3H-arginine uptake in control and ARG1/AGMAT-expressing SNU-449 cells with or without pre-loading with asparagine (Asn) or glutamine (Gln). N = 5–6. (B) Immunoblots of ARG1/AGMAT-expressing SNU-449 cells upon stable expression of ASNS or control. Calnexin serves as loading control. (C) Relative 3H-arginine uptake in control and ASNS-expressing SNU-449 ARG1/AGMAT-ex- pressing cells. N = 5. (D) Representative clonogenic growth assay of control and ASNS-expressing SNU-449 ARG1/ AGMAT-expressing cells grown in arginine- restricted medium. (E) mRNA levels of PSAT1, PSPH, GLSK, GLUT3, HK2, NNMT, and AOC3 in control and ASNS- expressing SNU-449 ARG1/AGMAT-expressing cells. N = 6–8. (F) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ASNS-expressing SNU-449 ARG1/ AGMAT-expressing cells. Calnexin serves as loading control. (G) mRNA levels of Asns in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV- shCtrl or AAV-shAsns. n = 6–7. (H) Number of macroscopic tumors per liver in L-dKO mice injected with AAV-shCtrl or AAV- shAsns. n = 7. (I) Arginine content in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-shCtrl or AAV-shAsns. n = 4–6. n.s. = not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t test (A, C, E, G, and H) and one-way ANOVA (I).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER L-Arginine-coupled agarose beads gbiosciences Cat#GENO786-1361 L-leucine-coupled agarose beads gbiosciences Cat#GENO786-1370 Critical commercial assays Total Polyamine Assay Kit BioVision Cat#K475 L-arginine ELISA kit MyBiosource Cat#MBS728648-96 Nano-Glo dual luciferase reporter assay kit Promega Cat#N1610 Deposited data HCC patients’ RNA sequencing data Ng et al., 202250 EGAS00001005073, EGAS00001005074 HCC patients’ proteome data Ng et al., 202250 PRIDE (PXD025705, PXD025836) Mouse RNA sequencing data Dimitrakopoulos et al., 202125 SRP156216 Mouse proteome data Dimitrakopoulos et al., 202125 https://github.com/cbgethz/netics/tree/ master/mouse_data Mouse and HCC patients’ metabolomics This paper MASSIVE: MSV000092406 RNA sequencing data of ARG1/AGMAT-expressing (and control) and RBM39-depleted (and control) SNU-449 cells This paper GEO: PRJNA940402 Mouse, HCC patients, and SNU-449 proteome data of pulldowns using Leu- and Arg-coupled agarose beads This paper MASSIVE: MSV000091516 Experimental models: Cell lines SNU-182 Diego Calvisi (University of Sassari, Italy) N/A SNU-449 Diego Calvisi (University of Sassari, Italy) N/A HLE Diego Calvisi (University of Sassari, Italy) N/A PLC Diego Calvisi (University of Sassari, Italy) N/A Hep40 Diego Calvisi (University of Sassari, Italy) N/A Hep3B Diego Calvisi (University of Sassari, Italy) N/A Huh6 Diego Calvisi (University of Sassari, Italy) N/A Huh7 Diego Calvisi (University of Sassari, Italy) N/A HepG2 Diego Calvisi (University of Sassari, Italy) N/A SNU-475 Gerhard Christofori (University of Basel, Switzerland) N/A SNU-423 Gerhard Christofori (University of Basel, Switzerland) N/A Huh1 Gerhard Christofori (University of Basel, Switzerland) N/A HEK293 ATCC CRL-1573 HEK239T ATCC CRL-3216 SNU-449 control This paper N/A SNU-449 ARG1 This paper N/A SNU-449 AGMAT This paper N/A SNU-449 ARG1/AGMAT This paper N/A SNU-449 ASS1-FLAG This paper N/A SNU-449 ARG1/AGMAT +ASS1 This paper N/A SNU-449 ARG1/AGMAT +3xHA-ASS1 This paper N/A Huh7 sgCtrl This paper N/A Huh7 sgARG1 This paper N/A Huh7 sgAGMAT This paper N/A SNU-449 ARG1/AGMAT +control (for ASNS) This paper N/A (Continued on next page) ll OPEN ACCESS Cell 186, 5068–5083.e1–e12, November 9, 2023 e2 Article

Techniques: Control, Expressing, Western Blot, Growth Assay, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 7. ARG1, AGMAT, arginine, and RBM39 in human HCC patients (A) Schematic representation of arginine and polyamine metabolism in HCC patients. Boxes below enzymes indicate changes in mRNA (left box) and protein (right box) levels in human HCC tumors (T) compared to paired non-tumor (NT) biopsies, respectively. Color coding according to level of log2 fold-change as indicated. ‘‘?’’ indicates unknown identity. Tumor aggressiveness is indicated by Edmondson-Steiner grade low (Edm. low, grade I and II) and high (Edm. high, grade III and IV). n = 73 (Edm. low) and n = 49 (Edm. high) for mRNA; n = 30 (Edm. low) and n = 21 (Edm. high) for protein. (B) Immunoblots of ARG1, AGMAT, RBM39, and ASNS in paired non-tumor (NT) and tumor (T) tissues of five HCC patients. Calnexin serves as loading control. (C) Tissue microarray for ARG1 and AGMAT. ARG1, normal liver n = 58, HCC n = 160; AGMAT, normal liver n = 49, HCC n = 142. (D) Representative IHC of ARG1 and AGMAT of an HCC patient (from C). Non-tumor, NT; tumor, T.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER L-Arginine-coupled agarose beads gbiosciences Cat#GENO786-1361 L-leucine-coupled agarose beads gbiosciences Cat#GENO786-1370 Critical commercial assays Total Polyamine Assay Kit BioVision Cat#K475 L-arginine ELISA kit MyBiosource Cat#MBS728648-96 Nano-Glo dual luciferase reporter assay kit Promega Cat#N1610 Deposited data HCC patients’ RNA sequencing data Ng et al., 202250 EGAS00001005073, EGAS00001005074 HCC patients’ proteome data Ng et al., 202250 PRIDE (PXD025705, PXD025836) Mouse RNA sequencing data Dimitrakopoulos et al., 202125 SRP156216 Mouse proteome data Dimitrakopoulos et al., 202125 https://github.com/cbgethz/netics/tree/ master/mouse_data Mouse and HCC patients’ metabolomics This paper MASSIVE: MSV000092406 RNA sequencing data of ARG1/AGMAT-expressing (and control) and RBM39-depleted (and control) SNU-449 cells This paper GEO: PRJNA940402 Mouse, HCC patients, and SNU-449 proteome data of pulldowns using Leu- and Arg-coupled agarose beads This paper MASSIVE: MSV000091516 Experimental models: Cell lines SNU-182 Diego Calvisi (University of Sassari, Italy) N/A SNU-449 Diego Calvisi (University of Sassari, Italy) N/A HLE Diego Calvisi (University of Sassari, Italy) N/A PLC Diego Calvisi (University of Sassari, Italy) N/A Hep40 Diego Calvisi (University of Sassari, Italy) N/A Hep3B Diego Calvisi (University of Sassari, Italy) N/A Huh6 Diego Calvisi (University of Sassari, Italy) N/A Huh7 Diego Calvisi (University of Sassari, Italy) N/A HepG2 Diego Calvisi (University of Sassari, Italy) N/A SNU-475 Gerhard Christofori (University of Basel, Switzerland) N/A SNU-423 Gerhard Christofori (University of Basel, Switzerland) N/A Huh1 Gerhard Christofori (University of Basel, Switzerland) N/A HEK293 ATCC CRL-1573 HEK239T ATCC CRL-3216 SNU-449 control This paper N/A SNU-449 ARG1 This paper N/A SNU-449 AGMAT This paper N/A SNU-449 ARG1/AGMAT This paper N/A SNU-449 ASS1-FLAG This paper N/A SNU-449 ARG1/AGMAT +ASS1 This paper N/A SNU-449 ARG1/AGMAT +3xHA-ASS1 This paper N/A Huh7 sgCtrl This paper N/A Huh7 sgARG1 This paper N/A Huh7 sgAGMAT This paper N/A SNU-449 ARG1/AGMAT +control (for ASNS) This paper N/A (Continued on next page) ll OPEN ACCESS Cell 186, 5068–5083.e1–e12, November 9, 2023 e2 Article

Techniques: Western Blot, Control, Microarray

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy.

doi: 10.1073/pnas.0911220107

Figure Lengend Snippet: Fig. 1. LRP6 expression is frequently up-regulated in a subset of human breast cancer tissues and cell lines. (A) Breast cancer TissueScan Real-Time qPCR array, containing seven normal/Stage 0 cDNAs and 41 human breast cancer cDNAs, was analyzed for LRP6 expression by real-time PCR. Averages of relative LRP6 expression from three independent plates are plotted with clinical status indicated. LRP6 mRNA levels are markedly up-regulated in a subset of human breast cancer tissues. #Samples with elevated HER2 tran- scripts. (B and C) Breast cancer tissue microarray was used for IHC staining of LRP6. (B) Representatives of LRP6 staining in normal and malignant breast tissue are shown. LRP6 antibody (C-term T1546, Abgent), which specifically recognizes human LRP6, was used for IHC staining. (C) The quantification of LRP6 IHC staining was determined from three independent experiments. Staining intensity was scored as absent (0), weak (1), moderate (2), or strong (3). Four observations were made on each slide by independent inves- tigators, and a mean score was recorded. (D) Expression of LRP6 in human mammary epithelial cell (MCF-10A) and indicated breast cancer cell lines analyzed by Western blot analysis. *P < 0.05; **P < 0.01.

Article Snippet: MDA-MB-231, MDA-MB-157, SKBR3, MCF-7, MDA-MB-435s, MDA-MB-361, HCC1187, HCC1143, HCC1806, HCC38, HCC1937, HCC1395, T-47D, and CAMA1 breast cancer cell lines and MCF-10A nontransformed cells were all from the American Type Culture Collection (ATCC) and grown according to ATCC recommendations.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Microarray, Immunohistochemistry, Staining, Western Blot

Journal: Therapeutic Advances in Musculoskeletal Disease

Article Title: Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma

doi: 10.1177/1759720X21995069

Figure Lengend Snippet: Expression of CDK7 in osteosarcoma cell lines, osteosarcoma patient fresh tissues, and osteosarcoma tissue microarray (TMA). (a) Expression levels of CDK7 protein in osteosarcoma cell lines (U2OS, KHOS, 143B, Saos-2, MG63, and MNNG/HOS) were higher than the expression of CDK7 in the normal osteoblast cell line (HOB-c, NHOst) as measured by Western blot. (b) Densitometry quantification of the Western blots of CDK7 from (a), presented as relative to tubulin expression. The data represent the mean ± SE of the experiment carried out in triplicate. (c) CDK7 expression in seven human osteosarcoma fresh tissues measured by Western blot. (d) Densitometry quantification of the Western blots of CDK7 from (c), presented as relative to tubulin expression. The data represent the mean ± SE of the experiment carried out in triplicate. (e) Representative images of different immunohistochemistry staining intensities of CDK7 and HE staining in an osteosarcoma TMA are shown in osteosarcoma tissues. Based on the CDK7 staining intensity within the tumor samples, the staining patterns were divided into six groups: no positive staining (0); <10% positive cells (1+); 10–25% positive cells (2+); 26–50% positive cells (3+); 51–75% positive cells (4+); >75% positive cells (5+). (Original magnification, 400×; scale bar, 50 µm). (f) Tumors with the staining score of ⩽2+ were defined as the low CDK7 expression group, ⩾3+ were defined as the high CDK7 expression group. Pie chart representing relative frequency of different CDK7 expression levels in osteosarcoma TMA. CKD7, cyclin-dependent kinase 7.

Article Snippet: The human osteosarcoma cell lines U2OS, Saos-2, MG63, MNNG/HOS, and 143B were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Microarray, Western Blot, Immunohistochemistry, Staining

Journal: Therapeutic Advances in Musculoskeletal Disease

Article Title: Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma

doi: 10.1177/1759720X21995069

Figure Lengend Snippet: Effects of the CDK7 inhibitor BS-181 on the activity of CDK7 and cell growth in osteosarcoma cell lines. BS-181, at the indicated concentrations, inhibited osteosarcoma cell proliferation in (a) cell growth and proliferation of KHOS, and (b) U2OS cell lines, which was determined by MTT assays after treatment with BS-181 for 6 days. The data represent the mean ± SE of two experiments carried out in triplicate. (c) Microscopy images of morphologic changes and a reduction in cell number after 72 h of BS-181 treatment (scale bar, 100 µm). (d) The expression of proteins involved in the CDK7-signaling pathway in the KHOS osteosarcoma cell line was examined by Western blot after 48 h of BS-181 treatment. (e) Semiquantitative analysis of (d) densitometry relative to tubulin. The data represent the mean ± SE of the experiment carried out in triplicate. (f) The expression of proteins involved in the CDK7-signaling pathway in the U2OS osteosarcoma cell line was examined by Western blot after 48 h of BS-181 treatment. (g) Semiquantitative analysis of (f) densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate. CKD7, cyclin-dependent kinase 7; RNAPII, RNA polymerase II.

Article Snippet: The human osteosarcoma cell lines U2OS, Saos-2, MG63, MNNG/HOS, and 143B were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Activity Assay, Microscopy, Expressing, Western Blot

Journal: Therapeutic Advances in Musculoskeletal Disease

Article Title: Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma

doi: 10.1177/1759720X21995069

Figure Lengend Snippet: CDK7 inhibition reduced osteosarcoma cell clonogenicity in vitro and decreased the spheroid diameter of osteosarcoma cell lines in a three-dimensional (3D) cell culture. (a) Representative results of colony formation in KHOS and U2OS. The numbers of colonies and their sizes were markedly decreased in cells treated with BS-181. (b, c) Representative images of KHOS and U2OS were measured after 10 µM of BS-181 treatment in a 3D cell culture. Spheroid formation of (b) KHOS and (c) U2OS were significantly smaller than untreated cells at all observation points. Cell fluorescence images of spheroid formation were taken after 12 days of cultivation (scale bar, 100 µm). (d, e) The average relative spheroid diameter of (b) KHOS and (c) U2OS treated with CDK7 inhibitor compared with untreated cells at an observation point of 12 days. The data are mean ± SE of the two experiments carried out in triplicate (** indicates p < 0.001). CKD7, cyclin-dependent kinase 7.

Article Snippet: The human osteosarcoma cell lines U2OS, Saos-2, MG63, MNNG/HOS, and 143B were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Inhibition, In Vitro, Cell Culture, Fluorescence

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Structures and cytotoxic activities of magnolol, M2M, and MM1. ( a ) Chemical structures of magnolol, M2M, and MM1. ( b and c ) A375 and A431 skin cancer cell lines were treated with various concentrations of magnolol, M2M, or MM1 for 24 h. Their cytotoxic effects and the viabilities of the treated cells were detected with ( b ) SRB assays and ( c ) MTT assays. ( d ) Flow cytometric analysis of cell cycle. A375 and A431 cells were treated with 75 μ M of magnolol (Mag) or MM1 for 24 h. The treated cells were stained with propidium iodide and analyzed by flow cytometry. The percentages of cells in the sub-G1 region (M1) are indicated. Data shown in b and c are expressed as the mean±S.D. of two independent experiments. Symbols: * P <0.05; ** P <0.01; and *** P <0.001, as analyzed by unpaired t -tests. Data shown in d are from one of two similar results

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Effects of magnolol and MM1 on the clonogenic ability and anchorage-independent growth of skin cancer cells. ( a ) A375 (left panel) and A431 (right panel) cells were treated with various concentrations of magnolol or MM1 for 6 days and then cultured for an additional 8 days in the absence of drugs. The numbers of foci were scored, and the data are presented as the relative focus-forming ability (FFA). ( b ) A375 cells were treated with different concentrations of magnolol and MM1. The anchorage-independent growth was assessed as described in the Materials and Methods section. Data are expressed as the mean±S.D. of three independent experiments. Symbols: * P <0.05, as analyzed by unpaired t -tests

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Cell Culture

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Effect of magnolol and MM1 on the induction of apoptosis in A375 cells. ( a ) A375 cells were treated with magnolol or MM1 for 24 h, and western blotting was used to assess cell lysates for the cleavages of PARP and procaspase-7, -8, and -9. ( b and c ) Effect of caspase inhibition on the apoptosis of MM1-treated A375 cells. A375 cells were treated with 75 μ M MM1 in the presence or absence of the broad-spectrum caspase inhibitor, z-VAD-fmk (40 μ M), for 24 h. Phase-contrast images of MM1-treated cells treated with or without z-VAD-fmk are shown in b . Western blot analysis was used to assess cell lysates for the cleavages of procaspase-7, -8, and 9, with β -actin used as a loading control ( c )

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Inhibition, Control

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Effects of magnolol and MM1 on the growth of xenograft tumors in nude mice. A375 cells were injected subcutaneously into the flank of each mouse. When the tumor volumes reached about 50 mm 3 , the mice were i.p. injected with magnolol (1 mM in 100 μ l acetone), MM1 (1 mM in 100 μ l acetone), or DMSO (control) three times per week ( n =6 per group). The tumor volume ( a ) was determined twice weekly, while body weight ( b ) was measured daily. The xenograft tumors were excised from the mice at the end of experiment in 15 days. The sizes of representative tumors excised from the different groups are shown at the top of a , while hematoxylin–eosin staining (magnification, × 200, upper panel) and IHC staining for activated caspase-3 (magnification, × 200, lower panel) are shown in c . The results shown in a and b are presented as the means±S.D. of six mice. Symbols: * P <0.05 by unpaired t -tests

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Injection, Control, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: MM1 upregulates lncRNA GAS5. A375 cells were treated with or without 50 μ M MM1 for 24 h, and total RNA was subjected to microarray analysis as described in the Materials and Methods section. The heatmap shown in a presents a hierarchical clustering of the transcriptome profiles from duplicate cultures of untreated and MM1-treated A375 cells. The relative expression of each RNA transcript is indicated by color, and ranges from red (higher expression) to green (lower expression). ( b ) Functional classification of the differentially expressed genes in MM1-treated A375 cells, as assessed using the DAVID Database web server. The differentially expressed proteins are linked to a number of biological processes. ( c ) Upregulation of lncRNA GAS5 in magnolol- or MM1-treated A375 cells. A375 cells were treated with the indicated concentrations of magnolol or MM1 for 24 h, and the expression levels of lncRNA GAS5 were determined by real time RT-PCR, as described in the Materials and Methods section. The expressions of lncRNA GAS5 in the magnolol- and MM1-treated cells were normalized to that of the untreated cells, and are presented as relative expression levels. The data shown represent the mean±S.D. of three independent experiments. Symbol: ** P <0.01, as analyzed with the unpaired t -test

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Microarray, Expressing, Functional Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: List of the 20 highest-scored lncRNA differentially expressed in MM1-treated A375 cells

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: lncRNA GAS5 plays a critical role in the MM1-mediated induction of apoptosis. ( a ) Effect of lncRNA GAS5 depletion on cell apoptosis. Left panel: A375 cells were transfected with siRNA against lncRNA GAS5 (si-lncRNA GAS5) at 50 nM or with nontargeting siRNA(−). After 48 h, the transfected cells were treated with 75 μ M of MM1 for 24 h. The levels of lncRNA GAS5 were assessed by real-time RT-PCR and expressed as relative to that of non-MM1-treated control (Left panel). The cleavage of PARP and caspase-9 was analyzed by western blotting (Right panel). β -actin served as an internal control. ( b and c ) Effect of lncRNA GAS5 overexpression on cell proliferation and apoptosis. A375 cells were transfected with pCDNA3.1-lncRNA GAS5 or the empty vector (control). After 48 h, the transfected cells were assayed for expression of lncRNA GAS5 ( b , left panel), for viability by MTT method ( b , right panel), and for the induction of apoptosis by phosphatidylserine exposure with Annexin V-FITC using flow cytometry ( c ). Data are expressed as the mean±S.D. of three independent experiments. Symbols: ** P <0.01; and *** P <0.001, as analyzed by unpaired t -tests

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Transfection, Quantitative RT-PCR, Control, Western Blot, Over Expression, Plasmid Preparation, Expressing, Flow Cytometry

Journal: Cell death & disease

Article Title: Interleukin-33 (IL-33) promotes DNA damage-resistance in lung cancer.

doi: 10.1038/s41419-025-07624-x

Figure Lengend Snippet: Fig. 3 IL-33 in the TME promotes chemoresistance of tumor cells. A Kaplan-Meier survival curve illustrating overall survival (OS) data from lung cancer patients. B The comparison of IL-33 protein expression between normal and cancerous tissues was conducted using IHC staining on a tissue microarray composed of 90 lung adenocarcinoma samples along with normal lung tissues. C Quantitative analysis of IL-33 protein expression levels in tissue microarrays was conducted using ImageJ. D A Kaplan−Meier survival curve was generated to illustrate the OS data of lung cancer patients represented in the tissue microarray. E Schematic diagram depicting the co-culture experiments. F A549 cells, either in single culture or co-cultured with CAFs, were treated with 3 μM CDDP or in combination with IL-33 neutralizing antibody for 48 h, and then cells were harvested for IB analysis. (G) A549 cells, either in single culture or co-cultured with CAFs, were treated with 1 μM Dox or in combination with IL-33 neutralizing antibody for 48 h, and then cells were harvested for IB analysis. H A549 cells were treated with 1 μM Dox or in combination with IL-33 recombinant protein for 48 h, and then cells were harvested for IB analysis. I A549 cells were treated with 1 μM Dox or in combination with IL-33 recombinant protein for 48 h and then cells were harvested for IF staining. J A549 cells were treated with 1 μM Dox or in combination with IL-33 protein for 48 h, and then cells were harvested for alkaline comet assay.

Article Snippet: Cell culture, virus infection, plasmid transfection A549 and H1299 cells were obtained from the American Type Culture Collection (ATCC, RRID: CVCL_0023 and RRID: CVCL_B7N8) and A549-DDP cells were obtained from Guangzhou Cellcook Biotech Co., Ltd.

Techniques: Comparison, Expressing, Immunohistochemistry, Microarray, Generated, Co-Culture Assay, Cell Culture, Recombinant, Staining, Alkaline Single Cell Gel Electrophoresis

Journal: Cell death & disease

Article Title: Interleukin-33 (IL-33) promotes DNA damage-resistance in lung cancer.

doi: 10.1038/s41419-025-07624-x

Figure Lengend Snippet: Fig. 4 IL-33 overexpression reduces the efficacy of chemotherapeutic drugs in tumor cells. A Schematic representation of IL-33 full-length and IL-33 cytokine domain constructs. B IB analysis was performed using virus-infected A549 cells, including control (Ctrl), IL-33-F OE, and IL-33-C OE groups. C Detection of IL-33 in the cell culture supernatants of A549 cells (Ctrl, IL-33-F OE, and IL-33-C OE) was performed using ELISA. D Virus- infected A549 cells (Ctrl, IL-33-F OE, and IL-33-C OE) were treated with or without 1 μM Dox for 48 h, and then cells were harvested for IB analysis and quantification of IB band density using ImageJ. E Virus-infected A549 cells (Ctrl, IL-33-F OE, and IL-33-C OE) were treated with or without 1 μM Dox for 48 h, and then cells were harvested for IF staining. F Virus-infected A549 cells (Ctrl, IL-33-F OE, and IL-33-C OE) were treated with or without 3 μM CDDP for 48 h, and then cells were harvested for IF staining. G Virus-infected A549 cells (Ctrl, IL-33-F OE, and IL-33-C OE) were treated with or without 1 μM Dox for 48 h, and then cells were harvested for alkaline comet assay. H Virus-infected A549 cells (Ctrl, IL-33-F OE, and IL-33-C OE) cells were treated with different concentrations of CDDP as indicated for 72 h, and the viability of cells was determined by the MTT assay. I Cell lysates from Control A549 cells and DDP were separated using a nucleus-cytoplasm separation kit and subjected to IB.

Article Snippet: Cell culture, virus infection, plasmid transfection A549 and H1299 cells were obtained from the American Type Culture Collection (ATCC, RRID: CVCL_0023 and RRID: CVCL_B7N8) and A549-DDP cells were obtained from Guangzhou Cellcook Biotech Co., Ltd.

Techniques: Over Expression, Construct, Virus, Infection, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Alkaline Single Cell Gel Electrophoresis, MTT Assay

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hypoxia-associated circRNA profiling and expression characteristics of Hsa_circ_0000566 in osteosarcoma (OS). (A) CircRNA microarray analysis reveals 35 upregulated and 23 downregulated circRNAs in OS cells under normoxic and hypoxic conditions. The black arrow represents Hsa_circ_0000566. (B) OS cells incubated under various oxygen concentrations. Total RNA extraction was performed for qRT-PCR assay. Western blotting was performed to determine the protein level of HIF-1α. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Scale bars, 200 μm. (C) Hsa_circ_0000566 expression is much higher in primary OS tissue than in chondroma tissue. Results are representative images according to three different experiments. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) results comparing Hsa_circ_0000566 mRNA expression in 12 OS and chondroma samples. Results are reported as mean ± SD, *p < 0.05, n = 12. (E) Hsa_circ_0000566 expression levels in hFOB1.19 and various OS cell lines. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Schematic diagram showing Hsa_circ_0000566 back-spliced by exons 2-11 of the VRK1 gene and the corresponding Sanger sequencing. (G) RT-PCR results validating the presence of Hsa_circ_0000566 in 143B and HOS cells. Various primers amplified the Hsa_circ_0000566 region in cDNA but not in genomic DNA. β-actin was used as the negative control. Divergent primers are presented as the opposite direction of the arrowhead, and the convergent primers were shown as the face-to-face direction of the arrowhead. (H) RT-PCR results indicating Hsa_circ_0000566 and VRK1 mRNA expression in untreated 143B and HOS cells and in the cells subjected to treatment with RNase-R. (I) RNA fluorescence in situ hybridization (FISH) results revealing Hsa_circ_0000566 localized mainly in the cytoplasm. Hsa_circ_0000566 probes were labeled with cy3 and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 μm. (J) qRT-PCR determination of the main localization of Hsa_circ_0000566 in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Microarray, Incubation, RNA Extraction, Quantitative RT-PCR, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Fluorescence, In Situ Hybridization, Labeling, Staining

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 contributes to in vitro osteosarcoma (OS) cell progression under hypoxic conditions. (A) Hsa_circ_0000566 overexpression and knockdown induced and repressed OS cell proliferation under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Circ_0000566 represents Hsa_circ_0000566 overexpression, and si circ_0000566 represents Hsa_circ_0000566 knockdown. Vector and Si NC represents the negative control of Hsa_circ_0000566 overexpression and Hsa_circ_0000566 knockdown, respectively. (B) EdU exhibits the impact of Hsa_circ_0000566 on OS cell proliferation under hypoxia. Nuclei are stained with 4’,6-diamidino-2-phenylindole (DAPI). Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (C) Colony formation experiment verifies Hsa_circ_0000566 functions in OS cells under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) Soft agar colony formation assay indicates the effects of Hsa_circ_0000566 on 143B and HOS cell colony forming capacity under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (E) OS cell migration capacity as determined by Transwell™ migration assays. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (F) Flow cytometry verifies Hsa_circ_0000566 functions in OS cell apoptosis. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, Over Expression, Knockdown, Standard Deviation, Plasmid Preparation, Negative Control, Staining, Soft Agar Assay, Migration, Flow Cytometry

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 accelerates osteosarcoma (OS) glucose metabolism and regulates hypoxia-enhanced glycolysis. (A) Colors of the media indicate that Hsa_circ_0000566 silencing decreased lactate accumulation under hypoxia. (B-C) Quantitative real-time polymerase chain reaction (qRT-PCR) or western blots evaluating the expression levels of genes involved in glucose metabolism in 143B and HOS cells transfected with Hsa_circ_0000566-overexpressing, Hsa_circ_0000566 (shRNA), or vector plasmids. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (D) Hsa_circ_0000566 knockdown in OS cells with decreased lactate accumulation, while Hsa_circ_0000566 overexpression has increased lactate accumulation. Results are reported as mean ± SD, *p < 0.05, n = 3. (E) Extracellular acidification rate (ECAR) indicates glycolysis rate. ECAR decreases in response to Hsa_circ_0000566 knockdown and increases in response to Hsa_circ_0000566 overexpression. Oxygen consumption rate (OCR) represented mitochondrial respiratory capacity. OCR is enhanced in response to Hsa_circ_0000566 silencing and reduced in response to Hsa_circ_0000566 overexpression in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Plasmid Preparation, Standard Deviation, Knockdown, Over Expression

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 establishes interactions with HIF-1α and confers protection against ubiquitination-mediating degradation. (A) Effects of Hsa_circ_0000566 knockdown and Hsa_circ_0000566 overexpression on mRNA and protein expression in 143B and HOS cells under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (B) Western blotting results revealing the impact of bortezomib treatment on the changes occurring at HIF-1α protein level mediated by Hsa_circ_0000566 silencing and vector transfection. (C) Western blotting assessment of the impact of CHX treatment on the variations in HIF-1α protein levels affected by Hsa_circ_0000566 silencing and vectors. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) The western blot illustrates the effects of Hsa_circ_0000566 knockdown in the Hyp564 HIF-1α protein levels in the presence or absence of bortezomib treatment. (E) Immunoprecipitation assessing the HIF-1α ubiquitination levels in Hsa_circ_0000566 silencing and Hsa_circ_0000566 overexpressing osteosarcoma (OS) cells under hypoxia. Culture media were supplemented with bortezomib (250 nM) for 6 h. (F) The combination of Hsa_circ_0000566 with HIF-1α confirmed by radioimmunoprecipitation (RIP). Results are reported as mean ± SD, *p < 0.05, n = 3. (G) Pulldown assay validation of the interaction between Hsa_circ_0000566 and HIF-1α. (H) A RIP assay of HIF-1α regions interacting with Hsa_circ_0000566. Schematic diagram shows HIF-1α protein fragments. Results are reported as mean ± SD, *p < 0.05, n = 3. (I) Interaction profile between Hsa_circ_0000566 and HIF-1α obtained from catRAPID (left). (J) Schematic diagram showing Hsa_circ_0000566 RNA fragments. Combinative regions between Hsa_circ_0000566 and HIF-1α were identified by RIP assay. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Ubiquitin Proteomics, Knockdown, Over Expression, Expressing, Standard Deviation, Western Blot, Plasmid Preparation, Transfection, Immunoprecipitation, Biomarker Discovery

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 promotes osteosarcoma (OS) glucose metabolism and tumorigenesis progression in vivo. (A) 143B cells stably transfected with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or empty vector plasmids. Nude mice were subcutaneously injected with 1 × 10 7 cells that were either stable negative controls or those with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or Hsa_circ_0000566 knockdown. Thirty days after injection, the animals were euthanized, and their tumors dissected and photographed. (B) Tumor weight measurements on the same day the mice were euthanized. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 5. (C) Tumor volumes (ab2/2) were calculated every 6 d from the day after the mice were injected with stable OS cells. (D-E) Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) exhibit the expression levels of the genes involved in glycolysis metabolism. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Fluorescence in situ hybridization (FISH), hematoxylin and eosin (H&E) staining, and immunohistochemistry (IHC) analysis indicate the OS organization in mice and relative GLUT1, GLUT4, PDK1, PDK4, and LDHA protein levels in tumors from different groups. (G) In situ tumor formation experiment reveals that HIF-1α overexpression recovered Hsa_circ_0000566 knockdown-induced tumor attenuation. Results are reported as mean ± SD, *p < 0.05, n = 4. (H) Micro-computed tomography (CT) indicates the functions of HIF-1α and Hsa_circ_0000566 knockdown in bone loss. (I) H&E staining of lung metastasis. In mice injected in the tail vein with various stable 143B cells, lung metastasis was detected using an in vivo bioluminescence imaging system. Results are reported as mean ± SD, *p < 0.05, n = 5.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vivo, Stable Transfection, Transfection, Knockdown, Over Expression, Plasmid Preparation, Injection, Standard Deviation, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Fluorescence, In Situ Hybridization, Staining, Immunohistochemistry, In Situ, Micro-CT, Imaging

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: LIN28A is overexpressed in human liver cancer. (A) Quantitative RT-PCR analysis of LIN28A mRNA levels in primary human hepatocytes (PHH) ( n = 10) as compared with human HCC cell lines (PLC ( n = 9), Hep3B ( n = 10), HepG2 ( n = 11)). (B,C) Quantitative RT-PCR analysis (A) and representative Western blot images depicting LIN28A mRNA and recombinant protein expression, respectively, after transfection of HepG2 cells (low endogenous LIN28A expression as compared with PLC and Hep33B) applying a LIN28A-overexpression (OE) plasmid vector (provided by G. Meister and described in detail elsewhere ( n = 4). (D) Immunofluorescence analysis of LIN28A expression and localization in two different HCC cell lines (PLC ( n = 2), Hep3B ( n = 2)) (representative images, 40-fold magnification). (E) LIN28A mRNA levels (qRT-PCR analysis) of paired human HCC and corresponding nontumorous liver tissue (CNTLT) samples ( n = 10pairs). (F-I) Immunohistochemical analysis of LIN28A protein expression and cellular localization patterns in patients applying tissue micro array derived paired HCC tissues and CNTLT. Staining intensity and percentage of positive cells were incorporated into a semiquantitative score describing "0" (very low/no expression), "1" (low/moderate expression) and "2" (strong expression). (F) depicts representative immunohistochemical images. (G) LIN28A protein expression score in human HCC tissues and CNTLT applying a tissue micro array (paired analyses). (H) LIN28A expression in HCC ( n = 103) as compared with CNTLT ( n = 115) (semiquantitative analysis applying Fisher's exact test). (I) Spearman correlation analysis of LIN28A expression levels in HCC patients with early (stage I & II) ( n = 71) compared with late (stage III and IV) tumor stages (UICC 2010). Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B), paired t-test (E,G), by two-sided Fisher's exact test (H), and Spearman correlation (I). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Quantitative RT-PCR, Western Blot, Recombinant, Expressing, Transfection, Over Expression, Plasmid Preparation, Immunofluorescence, Immunohistochemical staining, Microarray, Derivative Assay, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: LIN28A is a novel target gene of miR-622 in HCC. (A) MicroRNA recognition elements (MRE) in the LIN28A 3’UTR represent potential miR-622-binding sites and were identified applying the "TargetScan 7.2" database. (B) Quantitative RT-PCR analysis of LIN28A mRNA levels in control-transfected as compared to miR-622-transfected different HCC cell lines (PLC ( n = 3) ; Hep3B ( n = 3)). (C) Luciferase LIN28A 3′UTR-reporter (containing a conserved miR-622 MRE) activity in control-miR (CTR) as compared with miR-622-mimic (622)-transfected HCC cells (PLC) ( n = 3 ). (D) Schematic image depicting the pre-let-7-f-1 loop region (as predicted applying RNA-fold with the highlighted consensus motif for recognition and binding of LIN28 to pre-let-7-f-1 which was identified previously . (E) Schematic image depicting the pre-miR-622 stem loop (as predicted applying RNA-fold and miRNAMap ) and magnification of the pre-miR-622 loop region containing the identic sequence motif as identified previously for recognition and binding of LIN28 to pre-let-7. (F) Hypothesis image: According to pre-let-7, LIN28A might also bind to the terminal loop of pre-miR-622 with subsequent ZCCHC11-mediated oligouridylation followed by degradation of pre-miR-622. (G) RNA-pulldown ( n = 2) depicting binding of LIN28A to the let-7-g hairpin (positive control). No binding was found applying a negative control miR without the potential consensus motif (miR-18b) as well as a miR-622 and a miR-622-mutated (mutated consensus motif) hairpin. Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B). * P < 0.05, *** P < 0.001.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Binding Assay, Quantitative RT-PCR, Control, Transfection, Luciferase, Activity Assay, Sequencing, Positive Control, Negative Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: ZCCHC11 is overexpressed in HCC. (A) Quantitative RT-PCR analysis of ZCCHC11 mRNA levels in primary human hepatocytes (PHH) ( n = 9) as compared with human HCC cell lines (PLC ( n= 10), Hep3B ( n= 6), HepG2 ( n= 15)). (B) Representative Western blot image and (densitometric) quantification of ZCCHC11 protein levels in PHH ( n = 3) as compared with human HCC cell lines (PLC ( n = 4), Hep3B ( n = 4), HepG2 ( n = 4)). (C) ZCCHC11 mRNA levels (qRT-PCR analysis) in paired human HCC tissue samples and corresponding nontumorous liver tissues (CNTLT) ( n = 32pairs). (D) Transcriptomics data of ZCCHC11 RNA expression levels in HCC ( n = 369) and nontumorous liver tissues ( n = 50) derived from TCGA-data applying the Gene Expression Profiling Interactive Analysis (GEPIA) database. (E) ZCCHC11 overexpression in HCC tissues ( n = 2) as compared with nontumorous liver tissues ( n = 2) applying the human proteinatlas database (representative images). (E-H) Confirmation of ZCCHC11 protein expression and cellular localization patterns in patients applying tissue micro array derived paired HCC tissues and CNTLT. Staining intensity and percentage of positive cells were incorporated into a semi-quantitative score describing "0" (very low/no expression), "1" (low/moderate expression) and "2" (strong expression). (H) Representative immunohistochemistry images and paired quantification of ZCCHC11 expression score (G) as well as semiquantitative analysis applying Fischer's exact test (H) in HCC as compared with CNTLT. Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B,D), paired t-test (G), and two-sided Fisher's exact test (H). * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Quantitative RT-PCR, Western Blot, RNA Expression, Derivative Assay, Gene Expression, Over Expression, Expressing, Microarray, Staining, Immunohistochemistry

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: ZCCHC11 reveals oncogenic functions in HCC. (A-C) HCC cell lines (Hep3B ( n = 2), PLC ( n = 2)) were transfected with a control si-RNA-pool or with a specific si-RNA-pool targeting ZCCHC11 for 48 h. (A) ZCCHC11 mRNA levels as determined by qRT-PCR analysis. (B) Migration as determined applying Boyden chamber cell migration assays. (C) Colony numbers as determined applying clonogenic assays. (D) Real-time cell proliferation analysis of HCC cells transfected with an si-RNA-Pool directed against ZCCHC11 or a control si-RNA-Pool ( n = 4). (E) Correlated ZCCHC11 and CDK2, CDK4, CDK12, CDK14, CDK17, CDK20 RNA expression levels (log 2 (TPM)), respectively, in HCC tissues. TCGA-derived data were used applying the Gene Expression Profiling Interactive Analysis (GEPIA) database. The data represent RNA expression levels (log 2 (TPM)) in HCC tissues. (F,G) TCGA-derived datasets deposited on the Gene Expression Profiling Interactive Analysis (GEPIA) database (F) or the ProteinAtlas database (G), respectively. "HR": Hazard ratio. Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B,C,D) and Pearson correlation analysis (E). Survival analysis was performed computationally applying log-rank testing and hazard ratio estimates (F) or applying log-rank testing (Mantel-Cox) (G). * P < 0.05, ** P < 0.01, ns: non-significant.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Transfection, Control, Quantitative RT-PCR, Migration, RNA Expression, Derivative Assay, Gene Expression

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: ZCCHC11 is regulated by miR-622 in HCC. (A) Immunohistochemical analysis and correlation of ZCCHC11 and LIN28A expression levels applying tissue micro arrays (staining intensity and percentage of positive cells for both ZCCHC11 and LIN28A were incorporated into a semi-quantitative score describing "0" (very low/no expression), "1" (low/moderate expression) and "2" (strong expression)) ( n = 102). (B) MicroRNA recognition elements (MRE) in the ZCCHC11 3’UTR represent potential miR-622-binding sites and were identified applying the "TargetScan 7.2" database. (C) Quantitative RT-PCR analysis of ZCCHC11 mRNA levels in control-transfected as compared to miR-622-transfected different HCC cell lines (PLC ( n = 2) ; Hep3B ( n = 3)). (D) Representative Western blot images and (densitometric) quantification of ZCCHC11 protein levels in control-transfected as compared to miR-622-transfected different HCC cell lines (PLC ( n = 2) ; Hep3B ( n = 2)). (E) Luciferase ZCCHC11 3′UTR-reporter (containing a conserved miR-622 MRE) activity in control-miR (CTR) as compared with miR-622-mimic (622)-transfected HCC cells (PLC) ( n = 3 ). (F,G) TCGA-derived datasets and the Gene Expression Profiling Interactive Analysis (GEPIA) database were used for overall survival analysis comparing high and low ZCCHC11/miR-622-ratios (F) or high and low miR-622/ZCCHC11-ratios (G), respectively in HCC patients. "HR": Hazard ratio. Data are presented as the mean ± SEM. Statistical significance was determined by two-sided Fisher's exact test together with Spearman correlation analysis (A), and 2-tailed, unpaired t-test (C,D,E). Survival analysis was performed computationally applying log-rank testing and hazard ratio estimates (F,G). * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Immunohistochemical staining, Expressing, Staining, Binding Assay, Quantitative RT-PCR, Control, Transfection, Western Blot, Luciferase, Activity Assay, Derivative Assay, Gene Expression