Journal: Genome Biology

Article Title: Highly recurrent CBS epimutations in gastric cancer CpG island methylator phenotypes and inflammation

doi: 10.1186/s13059-021-02375-2

Figure Lengend Snippet: CBS epimutations associate with CIMP in primary GCs. a Overlap of genes that are promoter hypermethylated and downregulated at the RNA level in the discovery GC cell line panel, TCGA-stomach adenocarcinoma [STAD] and Singapore [SG] cohorts ( β -value difference ≥ 0.3 and q -value < 0.05) [left panel]. Average promoter methylation β -values and gene expression of CBS gene in STAD according to CIMP subtypes (* P < 0.001, two-tailed Wilcoxon rank sum test, each CIMP group vs. non-CIMP group) [right panel]. b Immunohistochemistry of CBS in a normal human stomach with black arrow indicating cytoplasmic staining in epithelial cells. Control sections were not treated with the primary antibody. c Summary of CBS staining in 66 cases of matched normal and gastric adenocarcinomas (* P < 0.05, two-tailed Fisher’s exact test) [left panel] and an example of a matched normal vs. tumor case with a negative score [right panel]. d Summary of somatic and germline genetic alterations at CBS in STAD. PALP, pyridoxal-phosphate dependent enzyme domain; CBS, cystathionine beta-synthase domain; aa, amino acid

Article Snippet: Normal human stomach antrum and fundus tissue slides were purchased from Novus Biologicals (NBP2-30203, NBP2-30204).

Techniques: Methylation, Gene Expression, Two Tailed Test, Immunohistochemistry, Staining, Control

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Formoterol (FORM) suppressed EPO‐induced VSMC senescence in mouse aorta. A) GO enrichment for the intersection genes. B) KEGG enrichment for the intersection genes. C) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of γH2AX (red particles) and αSMA (specific for SMCs, green particles) in ApoE −/− mice receiving vehicle, EPO and EPO+ medium‐dose formoterol treatment, respectively (scale bars = 10 µm). D) Quantitative analysis of γH2AX/αSMC colocalization in (C) ( n = 6 per group). E) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of SIRT1 (green particles) and αSMA (specific for SMCs, red particles) in ApoE −/− mice receiving vehicle, EPO and EPO+medium‐dose formoterol treatment, respectively. (scale bars = 10 µm). F) Quantitative analysis of γH2AX/αSMC colocalization in (E) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, One‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Formoterol ameliorated aortic senescence via β2AR. A) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of β2AR (green particles) and αSMA (specific for SMCs, red particles) in the aorta of ApoE −/− mice (scale bars = 10 µm). B) Representative western blot analysis of protein expression of SIRT1 and P21 in VSMCs treated with vehicle, EPO, EPO+0.01 nmol mL −1 formoterol, EPO+0.1 nmol mL −1 formoterol, EPO+1 nmol mL −1 formoterol and EPO+10 nmol mL −1 formoterol, respectively. C,D) Quantitative analysis of western blot in (B) ( n = 6 per group). E) Representative sections of SA‐β‐gal staining of VSMCs transfected with β2AR siRNA (siβ2AR) or negative control siRNA (siNC) and treated with vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. (scale bars = 10 µm). F) Quantitative analysis of percentage of VSMCs with positive SA‐β‐gal staining in (E) ( n = 6 per group). G) Representative western blot analysis of protein expression of SIRT1, P21 in VSMCs transfected with β2AR siβ2AR or siNC and treated with vehicle, EPO and EPO + 0.1 nmol mL −1 formoterol, respectively. H‐I) Quantitative analysis of western blot in (G) ( n = 6 per group). J) Representative images of immunostaining of SIRT1 in VSMCs transfected with β2AR siβ2AR or siNC and treated with vehicle, EPO, and EPO+ 0.1 nmol mL −1 formoterol, respectively. K) Quantitative analysis of colocalization of dapi/SIRT1 in VSMCs in(G) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, Two‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Western Blot, Expressing, Staining, Transfection, Negative Control, Immunostaining, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: cAMP‐regulated the effect of formoterol on VSMC senescence. A) Quantification of intracellular cAMP levels in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. B) Representative images of SA‐β‐gal staining of VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. (scale bars = 10 µm). C) Quantitative analysis of SA‐β‐gal staining in (B) ( n = 6 per group). D) Representative western blot assay of SIRT1, P21 in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. E‐F) Quantitative analysis of protein expression of SIRT1, P21 protein levels by western blot in (D), ( n = 6 per group). G) Representative images of immunostaining of SIRT1 in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. H) Quantitative analysis of colocalization of dapi/SIRT1 in (G) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, Two‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Control, Staining, Western Blot, Expressing, Immunostaining, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Casitas B‐lineage lymphoma (CBL) was essential for EPO‐induced VSMC senescence. A) Representative immunofluorescent analysis of the colocalization (yellow particles) of CBL (red particles) and SIRT1 (green particles) in VSMC receiving vehicle and EPO treatment respectively (scale bars = 10 µm). B) Quantitative analysis of CBL/SIRT1 colocalization in (A) ( n = 6 per group). C) Representative western blot analysis of protein expression of SIRT1 and P21 in VSMC transfected with siCBL or negative control siRNA (siNC) and treated with vehicle and EPO respectively. D‐E) Quantitative analysis of western blot in (C) ( n = 6 per group). F) Representative sections of SA‐β‐gal staining of VSMC transfected with CBL siRNA (siCBL) or negative control siRNA (siNC) and treated with vehicle and EPO respectively (scale bars = 10 µm). G) Quantitative analysis of the percentage of VSMC with positive SA‐β‐gal staining in (F) ( n = 6 per group). ** p < 0.01, *** p < 0.001, unpaired two‐tailed Student's t‐tests with Welch's correction were applied in (B). The other data were analyzed via Two‐way ANOVA followed by the Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Western Blot, Expressing, Transfection, Negative Control, Staining, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Schematic diagram showing the mechanism of therapeutic effects of medium‐dose formoterol on EPO‐induced AAA. Formoterol binds to β2AR and activates cAMP, which increases SIRT1 protein expression, leading to suppressed VSMC senescence induced by EPO. In contrast, SIRT1 is downregulated by EPO via activation of CBL, resulting in aggravated VSMC senescence. Thus, medium‐dose formoterol attenuated EPO‐induced AAA via β2AR/cAMP/SIRT1 pathways, which provides a promising medication for the treatment of AAA.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Expressing, Activation Assay

Journal: Thoracic Cancer

Article Title: Tumor suppressor effect of an antibody on xenotransplanted sarcomatoid mesothelioma cells

doi: 10.1111/1759-7714.14591

Figure Lengend Snippet: Representative immunofluorescent staining of cultured mesothelioma cells using AX10 antibody (a), immunohistochemical staining (b), and secondary antibody‐drug conjugate assay in vitro (c). (a) AX10 immunoreactivity in MPM‐1, −2, and −3 cells, representing sarcomatoid, epithelioid, and biphasic type mesothelioma, respectively. All MPM‐1, −2, and −3 cells exhibited AX10 antibody immunoreactivity at the cell surface. The staining was analyzed using a Guava easyCyte cell analyzer and accompanying software to obtain a one‐parameter log histogram. (b) AX10 immunoreactivity in various mesothelioma tissue specimens. Weak or no AX10 immunoreactivity was detected in five out of 10 epithelioid mesothelioma tissues (a). One out of five biphasic mesotheliomas exhibited AX10 immunoreactivity in spindle sarcomatoid components (arrow) but weak immunoreactivity in epithelioid components (arrowhead) (b). Five out of six sarcomatoid mesothelioma tissues exhibited strong AX10 immunoreactivity (c). Little AX10 immunoreactivity was detected in normal human tissues. No significant AX10 immunoreactivity was detected in the lung (d) (pleural mesothelial cells; insert) tissue specimens. Weak AX10 immunoreactivity was detected in myofibrous cells in the uterus (e). We did not detect any significant AX10 immunoreactivity in the brain, liver, or kidney, whereas strong AX10 immunoreactivity was observed in a nonmelanocytic (hypomelanocytic) melanoma tissue sample that was supplementally included in the microarray (f) (staining without AX10 antibody; insert). (c) MPM‐1 sarcomatoid mesothelioma cells were incubated with AX10 at 10, 100, and 1000 ng/mL followed by incubation with anti‐murine IgG (Fc) antibody conjugated to duocarmycin. Representative staining with Annexin V‐PI is presented. Note the dose‐dependent Annexin V‐positive and PI‐negative apoptotic MPM‐1 cells in the presence of AX10 antibody

Article Snippet: Tissue microarrays composed of mesothelioma (Cat. No. MS801b) and Food and Drug Administration (FDA) normal organ tissue arrays (Cat. No. NBP2‐78057) were purchased from US Biomax and Novus Biologicals, respectively.

Techniques: Staining, Cell Culture, Immunohistochemical staining, In Vitro, Software, Microarray, Incubation

Journal: Thoracic Cancer

Article Title: Tumor suppressor effect of an antibody on xenotransplanted sarcomatoid mesothelioma cells

doi: 10.1111/1759-7714.14591

Figure Lengend Snippet: AX10 does not affect cell proliferation, but significantly decreases Matrigel invasion activity of MPM‐1 sarcomatoid mesothelioma cells in vitro. (a) Representative cell proliferation assay. At 24 h, the cell number was 1.80 ± 0.10 (mock) and 1.77 ± 0.06 (AX10). Respective numbers at 48 h were 2.40 ± 0.10 (mock) and 2.37 ± 0.12 (AX10), while at 72 h they were 3.90 ± 0.20 (mock) and 4.20 ± 0.61 (AX10). The data represent means ± SD from triplicate assays (Student's t ‐test, p > 0.5). (b) AX10 significantly reduced Matrigel invasion activity of MPM‐1 cells (Student's t ‐test, p < 0.01). The number of invading cells was 59.7 ± 7.02 (mock) and 10.3 ± 1.52 (AX10) at 24 h, and 210.7 ± 11.4 (mock) and 15.0 ± 3.00 (AX10) at 48 h. Data from triplicate assays are expressed as means ± SD ( n = 3). (c) Cells that migrated to the lower surface of the membrane are shown (48 h). Original magnification, ×100

Article Snippet: Tissue microarrays composed of mesothelioma (Cat. No. MS801b) and Food and Drug Administration (FDA) normal organ tissue arrays (Cat. No. NBP2‐78057) were purchased from US Biomax and Novus Biologicals, respectively.

Techniques: Activity Assay, In Vitro, Proliferation Assay, Membrane

Journal: Thoracic Cancer

Article Title: Tumor suppressor effect of an antibody on xenotransplanted sarcomatoid mesothelioma cells

doi: 10.1111/1759-7714.14591

Figure Lengend Snippet: Inhibitory effect of AX10 on MPM‐1 xenotransplanted sarcomatoid mesothelioma cell proliferation. (a) Inoculation of AX10 antibody delayed the growth of xenotransplanted MPM‐1 sarcomatoid mesothelioma tumors. On day 0, SCID‐NOD mice were subcutaneously implanted with MPM‐1 cells. The following day, day 3, the mice were administered AX10 antibody or vehicle only by intraperitoneal injection and weekly thereafter as indicated by arrows. Values are represented as means ± standard error for n = 5 mice. Statistical significance was measured by a two‐sided unpaired Student's t ‐test (* p < 0.01). (b) On day 42, the xenotransplanted tumors were excised to determine their weight. Total tumor weights are represented as means ± standard error for n = 5 mice. Statistical significance was measured by a two‐sided unpaired Student's t ‐test ( p < 0.01). (c) Gross and histological appearance of a representative xenotransplanted tumor. Arrowhead indicates the tumor without AX10 antibody, while the arrow indicates the small tumor remaining following weekly AX10 injection. Note the elimination of tumor cells, which were histologically replaced by regenerative muscle in mice inoculated with AX10 antibody. Scale bar indicates 100 μm

Article Snippet: Tissue microarrays composed of mesothelioma (Cat. No. MS801b) and Food and Drug Administration (FDA) normal organ tissue arrays (Cat. No. NBP2‐78057) were purchased from US Biomax and Novus Biologicals, respectively.

Techniques: Injection

Journal: The Journal of Physiology

Article Title: Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium‐driven bile uptake

doi: 10.1113/JP272774

Figure Lengend Snippet: A , mRNA expression of selected genes encoding bile acid transporters in human pancreatic stellate cells (hPSCs) and human hepatocytes. PCR products for the sodium–taurocholate cotransporting polypeptide (NTCP, slc10A1) and sodium independent transporters, OATP4A1 ( slco4A1 ) and OATP1B3 (slco1B3) , are present in human hepatocytes; hPSCs express slc10A1 and slco4A1 , but not slco1B3 . B , immunoblotting shows expression of NTCP in different tissues; lines from the left: mass marker, hPSC, four positive controls (human liver and mouse pancreas, liver and kidney), two negative controls (mouse spleen and lung). C , immunohistofluorescence (IHF) staining for NTCP in fixed mouse tissue sections: the pancreas (upper panel) and the liver (lower panel). From the left: DAPI (blue), NTCP (white), overlay images (white arrows indicate PSCs), and transmitted light images. Insets show corresponding staining in controls without primary antibody. D , double IHF staining for NTCP and the bradykinin receptor B2 (BDKRB2). From the upper left: DAPI (blue), NTCP (green), BDKRB2 (red), fluorescence overlay (PSCs are indicated by white arrows). E and F , the comparison of homological sequences of three Na + ‐dependent mouse transporters (NTCP, ASBT and SOAT) with immunogens used for generation of the antibodies for immunoblotting ( E ) and IHF ( F ). Red letters represent amino acids identical to those in the immunogen sequence; blue letters are similar amino acids, and black letters show amino acids that are different. Only mouse NTCP sequences share high sequence identity with the immunogens.

Article Snippet: Human liver tissue lysate was obtained from Novus Biologicals, Littleton, CO, USA.

Techniques: Expressing, Western Blot, Marker, Immunohistofluorescence, Staining, Fluorescence, Comparison, Sequencing