Journal: Biochemistry and biophysics reports

Article Title: CA10 is associated with HBV-related hepatocarcinogenesis.

doi: 10.1016/j.bbrep.2022.101303

Figure Lengend Snippet: Fig. 1. CA10 overexpression is associated with sW182* non-sense mutation. CA10 expressions in sW182* NIH3T3 stable cells and its xenograft-derived cell line (W182-m1T) were compared with vector control or wild type by RT-PCR (A), Q-PCR (B) and western blotting (C), respectively. The CA10 expres sion in sW182* HepG2 stable cells was also compared with vector control or wild type by Q-PCR (D) and western blotting (E), respectively. (F) CA10 expres sions in the 48 HBV-related HCC tumor (T) and non- tumor tissues (N) from CGMH were analyzed by Q- PCR. Asterisks (*) indicate the tumor had HBS W182 non-sense mutation. Data are represented as mean ± SD from three independent experiments. Statistical significance was determined by Student’s unpaired t- test. **, P＜ 0.01.

Article Snippet: The primary antibodies used for western blotting were as follows: Bad (Rabbit Bad polyclonal antibody #9292 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-2 (Bcl-2 (D17C4) Rabbit monoclonal antibody #3498 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-xS/L (Bcl-xS/L (S-18) Rabbit polyclonal antibody: sc-634 (1:1000); Santa Cruz, CA, USA), Mcl-1 (Mcl-1 (D35A5) Rabbit monoclonal antibody #5453 (1:1000); Cell signaling, Beverly, MA, USA), CA10 (CA10 Rabbit polyclonal antibody: 12953-1-AP (1:500); Proteintech, Chicago, IL, USA), α-tubulin (α-tubulin Rabbit polyclonal antibody: ab18251 (1:10,000); Abcam, Cambridge, UK), and GAPDH (GAPDH Rabbit polyclonal antibody: GTX100118 (1:20,000); GeneTex, CA, USA).

Techniques: Over Expression, Mutagenesis, Derivative Assay, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Biochemistry and biophysics reports

Article Title: CA10 is associated with HBV-related hepatocarcinogenesis.

doi: 10.1016/j.bbrep.2022.101303

Figure Lengend Snippet: Fig. 2. Transforming activity study of CA10 overexpressing NIH3T3 cells. (A) CA10 overexpression in the stable cells were examined by western blotting. (B) Cell proliferation assays for vector control and CA10 overexpressing cells. (C, D) Clonogenic assays of CA10 overexpressing and vector control cells are shown in C and the statistical analysis is shown in D. The total number of colonies formed on each well were counted by AlphaEaseFC software. (E, F) Soft agar assays and the statistical analysis of CA10 overexpressing and vector control cells. Quan tifications of cell numbers or colony numbers for CA10 overexpressing cells are presented as fold change in comparison with vector control. Data are repre sented as mean ± SD from three independent experiments. Statistical signifi cance was determined by Student’s unpaired t-test. *, P＜ 0.05; **, P＜ 0.01; ***, P＜0.001.

Article Snippet: The primary antibodies used for western blotting were as follows: Bad (Rabbit Bad polyclonal antibody #9292 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-2 (Bcl-2 (D17C4) Rabbit monoclonal antibody #3498 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-xS/L (Bcl-xS/L (S-18) Rabbit polyclonal antibody: sc-634 (1:1000); Santa Cruz, CA, USA), Mcl-1 (Mcl-1 (D35A5) Rabbit monoclonal antibody #5453 (1:1000); Cell signaling, Beverly, MA, USA), CA10 (CA10 Rabbit polyclonal antibody: 12953-1-AP (1:500); Proteintech, Chicago, IL, USA), α-tubulin (α-tubulin Rabbit polyclonal antibody: ab18251 (1:10,000); Abcam, Cambridge, UK), and GAPDH (GAPDH Rabbit polyclonal antibody: GTX100118 (1:20,000); GeneTex, CA, USA).

Techniques: Activity Assay, Over Expression, Western Blot, Plasmid Preparation, Control, Software, Comparison

Journal: Biochemistry and biophysics reports

Article Title: CA10 is associated with HBV-related hepatocarcinogenesis.

doi: 10.1016/j.bbrep.2022.101303

Figure Lengend Snippet: Fig. 3. Migration/invasion and xenograft study of CA10 overexpressing cells. (A, B) Transwell migration assays of CA10 overexpressing cells or vector control. Photographs of 24-transwell insert membrane in a representative experiment are shown in A and the statistical analysis is shown in B. (C, D) Transwell invasion assays of CA10 overexpressing cells or vector control. Representative images are shown in C and the statistical analysis is shown in D. Quantification of migrated/invaded cell numbers for CA10 stable cells presented as fold change in comparison with vector control. Data are represented as mean ± SD from three independent experiments. Statistical sig nificance was determined by Student’s unpaired t- test. *, P＜ 0.05; **, P＜ 0.01; ***, P＜0.001. (E) Xenograft study of vector control. (F) Xenograft study of CA10 stable cells showed tumor formation in all four mice. Tumor volume was measured with a caliper and calculated by using the formula a × b2 × 0.5, where a and b represented the larger and smaller diameters, respectively.

Article Snippet: The primary antibodies used for western blotting were as follows: Bad (Rabbit Bad polyclonal antibody #9292 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-2 (Bcl-2 (D17C4) Rabbit monoclonal antibody #3498 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-xS/L (Bcl-xS/L (S-18) Rabbit polyclonal antibody: sc-634 (1:1000); Santa Cruz, CA, USA), Mcl-1 (Mcl-1 (D35A5) Rabbit monoclonal antibody #5453 (1:1000); Cell signaling, Beverly, MA, USA), CA10 (CA10 Rabbit polyclonal antibody: 12953-1-AP (1:500); Proteintech, Chicago, IL, USA), α-tubulin (α-tubulin Rabbit polyclonal antibody: ab18251 (1:10,000); Abcam, Cambridge, UK), and GAPDH (GAPDH Rabbit polyclonal antibody: GTX100118 (1:20,000); GeneTex, CA, USA).

Techniques: Migration, Plasmid Preparation, Control, Membrane, Comparison

Journal: Biochemistry and biophysics reports

Article Title: CA10 is associated with HBV-related hepatocarcinogenesis.

doi: 10.1016/j.bbrep.2022.101303

Figure Lengend Snippet: Fig. 4. Apoptosis-related protein expression profiles and the ER stress markers of CA10 overexpressing cells were examined by western blot analysis and RT- PCR, respectively. Apoptosis related signaling protein levels were measured by western blotting in the CA10 overexpressing stable cells (A) or CA10 knock-down stable clones: shCA10-1 and shCA10-2 (B). Band in tensity given underneath gel image was determined with ImageJ software, presented as fold change compared with controls, respectively. (C) Phosphor ylation of IRE1α was examined by western blotting. (D) ATF6 cleavage was evaluated by western blotting. (E) ATF4 expression was determined by western blotting. (F) Splicing of XBP-1 mRNA was examined by RT-PCR. The un-spliced and spliced forms of XBP-1 were indicated by arrows. GAPDH was used as a loading control.

Article Snippet: The primary antibodies used for western blotting were as follows: Bad (Rabbit Bad polyclonal antibody #9292 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-2 (Bcl-2 (D17C4) Rabbit monoclonal antibody #3498 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-xS/L (Bcl-xS/L (S-18) Rabbit polyclonal antibody: sc-634 (1:1000); Santa Cruz, CA, USA), Mcl-1 (Mcl-1 (D35A5) Rabbit monoclonal antibody #5453 (1:1000); Cell signaling, Beverly, MA, USA), CA10 (CA10 Rabbit polyclonal antibody: 12953-1-AP (1:500); Proteintech, Chicago, IL, USA), α-tubulin (α-tubulin Rabbit polyclonal antibody: ab18251 (1:10,000); Abcam, Cambridge, UK), and GAPDH (GAPDH Rabbit polyclonal antibody: GTX100118 (1:20,000); GeneTex, CA, USA).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Knockdown, Clone Assay, Software, Control

Journal: Biochemistry and biophysics reports

Article Title: CA10 is associated with HBV-related hepatocarcinogenesis.

doi: 10.1016/j.bbrep.2022.101303

Figure Lengend Snippet: Fig. 5. miR-27b targeted the 3′-untranslated region (3′-UTR) of CA10. (A) The CA10 promoter reporter plasmid was co-transfected with plasmids expressing wild type (WT) HBS or sW182* in HEK293 cells and Huh7 cells. Dual-Luciferase Reporter Assay system was performed for the promotor reporter assay. (B) miRNAs potentially targeting CA10 were predicted by the TargetScan, miRanda and PicTar. Venn diagram showed that there were only 4 candidate miRNAs (miR-27a, miR-27b, miR-128 and miR-185) selected by all 3 databases. (C) Q-PCR analysis of miR-27b expression in WT or sW182* NIH3T3 stable cells and its xenograft-derived cells (W182-m1T). (D) The miR-27b target region of human CA10 3′UTR. The mutant CA10 3′UTR consisting of mutations at the miR-27b binding site and red words indicate the mutated sequences. (E) Dual-Luciferase Reporter Assay of wild-type and mutant CA10-3′UTR while co- transfected with miR-27b at different ratios. Control treatment was cells only transfected with wild-type or mutant CA10-3′UTR reporter plasmid. Data was rep resented as mean ± SD from three independent ex periments. Statistical significance were determined by Student’s unpaired t-test. **, P＜ 0.01; ***, P＜ 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used for western blotting were as follows: Bad (Rabbit Bad polyclonal antibody #9292 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-2 (Bcl-2 (D17C4) Rabbit monoclonal antibody #3498 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-xS/L (Bcl-xS/L (S-18) Rabbit polyclonal antibody: sc-634 (1:1000); Santa Cruz, CA, USA), Mcl-1 (Mcl-1 (D35A5) Rabbit monoclonal antibody #5453 (1:1000); Cell signaling, Beverly, MA, USA), CA10 (CA10 Rabbit polyclonal antibody: 12953-1-AP (1:500); Proteintech, Chicago, IL, USA), α-tubulin (α-tubulin Rabbit polyclonal antibody: ab18251 (1:10,000); Abcam, Cambridge, UK), and GAPDH (GAPDH Rabbit polyclonal antibody: GTX100118 (1:20,000); GeneTex, CA, USA).

Techniques: Plasmid Preparation, Transfection, Expressing, Luciferase, Reporter Assay, Derivative Assay, Mutagenesis, Binding Assay, Control

Journal: Biochemistry and biophysics reports

Article Title: CA10 is associated with HBV-related hepatocarcinogenesis.

doi: 10.1016/j.bbrep.2022.101303

Figure Lengend Snippet: Fig. 6. CA10 overexpression in sW182* stable cells might be through repression of miR-27b. (A) Q-PCR analysis of miR-27b expression in WT or sW182* HepG2 stable cells. (B) Q-PCR analysis of miR27b expression in sW182* HepG2 stable cells after tran sient transfection with vector, miR-27b or anti-miR- 27b plasmid. (C) Q-PCR analysis of CA10 mRNA expression in sW182* HepG2 stable cells after tran sient transfection with vector, miR-27b or anti-miR- 27b plasmid. (D) Western blot analysis of CA10 expression in sW182* HepG2 stable cells after tran sient transfection with vector, miR-27b or anti-miR- 27b plasmid. (E) CA10 mRNA degradation was measured by real-time Q-PCR in sW182* HepG2 sta ble cells following transient transfection with vector or miR-27b plasmid. Data was represented as mean ± SD from three independent experiments. Statistical significance were determined by Student’s unpaired t- test. *, P＜ 0.05; **, P＜ 0.01.

Article Snippet: The primary antibodies used for western blotting were as follows: Bad (Rabbit Bad polyclonal antibody #9292 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-2 (Bcl-2 (D17C4) Rabbit monoclonal antibody #3498 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-xS/L (Bcl-xS/L (S-18) Rabbit polyclonal antibody: sc-634 (1:1000); Santa Cruz, CA, USA), Mcl-1 (Mcl-1 (D35A5) Rabbit monoclonal antibody #5453 (1:1000); Cell signaling, Beverly, MA, USA), CA10 (CA10 Rabbit polyclonal antibody: 12953-1-AP (1:500); Proteintech, Chicago, IL, USA), α-tubulin (α-tubulin Rabbit polyclonal antibody: ab18251 (1:10,000); Abcam, Cambridge, UK), and GAPDH (GAPDH Rabbit polyclonal antibody: GTX100118 (1:20,000); GeneTex, CA, USA).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: Biochemistry and biophysics reports

Article Title: CA10 is associated with HBV-related hepatocarcinogenesis.

doi: 10.1016/j.bbrep.2022.101303

Figure Lengend Snippet: Fig. 7. A schematic diagram illustrating induction of the oncogenic activity of CA10 through repression of miR-27b by HBS W182 non-sense mutation. sW182* could repress miR-27b expression and subsequently up-regulate CA10. Finally, CA10 overexpression could result in promotion of cell proliferation, cell migration and reduction of apoptosis by dysregulation of apoptosis-related proteins, suggesting that CA10 may play a role in hepatocarcinogenesis.

Article Snippet: The primary antibodies used for western blotting were as follows: Bad (Rabbit Bad polyclonal antibody #9292 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-2 (Bcl-2 (D17C4) Rabbit monoclonal antibody #3498 (1:1000); Cell signaling, Beverly, MA, USA), Bcl-xS/L (Bcl-xS/L (S-18) Rabbit polyclonal antibody: sc-634 (1:1000); Santa Cruz, CA, USA), Mcl-1 (Mcl-1 (D35A5) Rabbit monoclonal antibody #5453 (1:1000); Cell signaling, Beverly, MA, USA), CA10 (CA10 Rabbit polyclonal antibody: 12953-1-AP (1:500); Proteintech, Chicago, IL, USA), α-tubulin (α-tubulin Rabbit polyclonal antibody: ab18251 (1:10,000); Abcam, Cambridge, UK), and GAPDH (GAPDH Rabbit polyclonal antibody: GTX100118 (1:20,000); GeneTex, CA, USA).

Techniques: Activity Assay, Mutagenesis, Expressing, Over Expression, Migration

Journal: Neural Regeneration Research

Article Title: Unilateral rNurr1-V5 transgene expression in nigral dopaminergic neurons mitigates bilateral neuropathology and behavioral deficits in parkinsonian rats with α-synucleinopathy

doi: 10.4103/1673-5374.391190

Figure Lengend Snippet: Antibodies for immunohistochemistry staining

Article Snippet: The primary antibodies were mouse monoclonal anti-TH (1:1000; Sigma-Aldrich, Cat# T2928, RRID: AB_477569), rabbit polyclonal anti-TH (1:1000; Millipore, Temecula, CA, USA, Cat# AB5986, RRID: AB_92190), mouse monoclonal anti-V5 (1:500; Thermo Fisher Scientific, Cat# R960-25, RRID: AB_2556564), rabbit polyclonal anti-hCDNF (1:1000; ProSci, San Diego, CA, USA, Cat# 4343, RRID: AB_10909695), rabbit polyclonal anti-GFP (1:1500; Abcam, Cat# ab290, RRID: AB_303395), rabbit polyclonal anti-βIII-tubulin (1:300; Sigma-Aldrich, Cat# T2200, RRID: AB_262133), rabbit polyclonal anti-rNurr1 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-5568, RRID: AB_2267355), rabbit monoclonal anti-BDNF (1:400; Abcam, Cat# ab108319, RRID: AB_10862052, mouse monoclonal anti-LB-509 α-synuclein (1:500; Abcam, Cat# ab27766, RRID: AB_727020, mouse monoclonal anti-GFAP (Clone GA5; 1:500; Cell Signaling Technology, Cat# 3670, RRID: AB_561049, rabbit polyclonal anti-C3 (1:100; Abcam, Cat# ab11887, RRID: AB_298669), and rabbit polyclonal anti-S100A10 (1:100; Thermo Fisher Scientific, Cat# PA5-95505, RRID:AB_2807307).

Techniques: Immunohistochemistry

Journal: Neural Regeneration Research

Article Title: Unilateral rNurr1-V5 transgene expression in nigral dopaminergic neurons mitigates bilateral neuropathology and behavioral deficits in parkinsonian rats with α-synucleinopathy

doi: 10.4103/1673-5374.391190

Figure Lengend Snippet: Antibodies and fluorescent labels for double immunofluorescence assays

Article Snippet: The primary antibodies were mouse monoclonal anti-TH (1:1000; Sigma-Aldrich, Cat# T2928, RRID: AB_477569), rabbit polyclonal anti-TH (1:1000; Millipore, Temecula, CA, USA, Cat# AB5986, RRID: AB_92190), mouse monoclonal anti-V5 (1:500; Thermo Fisher Scientific, Cat# R960-25, RRID: AB_2556564), rabbit polyclonal anti-hCDNF (1:1000; ProSci, San Diego, CA, USA, Cat# 4343, RRID: AB_10909695), rabbit polyclonal anti-GFP (1:1500; Abcam, Cat# ab290, RRID: AB_303395), rabbit polyclonal anti-βIII-tubulin (1:300; Sigma-Aldrich, Cat# T2200, RRID: AB_262133), rabbit polyclonal anti-rNurr1 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-5568, RRID: AB_2267355), rabbit monoclonal anti-BDNF (1:400; Abcam, Cat# ab108319, RRID: AB_10862052, mouse monoclonal anti-LB-509 α-synuclein (1:500; Abcam, Cat# ab27766, RRID: AB_727020, mouse monoclonal anti-GFAP (Clone GA5; 1:500; Cell Signaling Technology, Cat# 3670, RRID: AB_561049, rabbit polyclonal anti-C3 (1:100; Abcam, Cat# ab11887, RRID: AB_298669), and rabbit polyclonal anti-S100A10 (1:100; Thermo Fisher Scientific, Cat# PA5-95505, RRID:AB_2807307).

Techniques: Immunofluorescence, Expressing, In Vitro, Activity Assay, Plasmid Preparation

Journal: NAR Genomics and Bioinformatics

Article Title: Joint single cell DNA-seq and RNA-seq of gastric cancer cell lines reveals rules of in vitro evolution

doi: 10.1093/nargab/lqaa016

Figure Lengend Snippet: Integrated single cell sequencing informs live-cell-sorting of NUGC-4 clones. ( A ) Phylogenetic tree of the four clones detected by scDNA-seq in NUGC-4. ( B ) G0/G1 cells are shown in tSNE space—every dot is a cell, and two clones of interest are highlighted in purple and green. The purple clone has three copies of a large genomic region on chromosome 2q (2q gain), whereas the green clone has an extra copy of that same segment (2q amplification). Cells are colored based on their CNVs, whereas their location in tSNE space is based on their expression signature. ( C ) The top four cell surface markers informing separation of the green and purple clone in tSNE space were: ANPEP, CXCR4, TM4SF4 and ITM2C. We used flow cytometry with these four markers to enrich for the respective clone. ( D ) Cytogenetic analysis of chromosome 2 identified three karyotypes among the two isolated subpopulations (top row), including a rearrangement on the q-arm (red arrow). All of the cells (100%) from the green subpopulation had a chromosome 2q rearrangement, whereas the purple subpopulation contained only 35% cells with the rearrangement.

Article Snippet: Antibodies used in this study include: anti-human CD13 (ANPEP) PeCy7 (clone WM15; BioLegend) 1:10, anti-human CD184 (CXCR4) BV421 (clone 12G, BioLegend) 1:10, anti-human TM4SF4 APC (R&D) 1:10, anti-human ITM2C (clone 2E8G11, Proteintech) 1:10.

Techniques: Sequencing, FACS, Clone Assay, Amplification, Expressing, Flow Cytometry, Isolation