Journal: Nature Communications

Article Title: Functional exploration of colorectal cancer genomes using Drosophila

doi: 10.1038/ncomms13615

Figure Lengend Snippet: ( a , b ) Plot of P -values indicating significance of compound rescue ( a ) and summary of compound response ( b ) of ras G12V and ras G12V p53 Ri pten Ri apc Ri animals. P -values ( a ) were obtained by comparing the dissemination phenotype after compound feeding to dimethyl sulfoxide (DMSO) fed flies (dissemination plots can be found in ). Blue dots represent statistically significant results. ( c ) Quantification of dissemination in ras G12V pten Ri and ras G12V p53 Ri apc Ri animals treated with BEZ235. ( d ) Western blot analysis of PI3K pathway output from hindguts with indicated genotypes 7 days after induction of transgenes and quantification. Syn, Syntaxin (loading control). ( e ) Time-course analysis of PI3K pathway activation status in control and ras G12V p53 Ri pten Ri apc Ri hindguts. ( f ) Western blot analysis of the biochemical response by ras G12V and ras G12V p53 Ri pten Ri apc Ri animals to PI3K pathway inhibitors. ( g ) Quantification of dissemination in indicated genotypes treated with BEZ235 or DMSO. ( h ) Schematic illustration of the mechanism of resistance to BEZ235: genetically activating mTORC1 promotes BEZ235 sensitivity. ( d , e ) Each data point represents the average response of two to five biological replicates with ten hindguts per replicate; error bars: s.e.m. ( a – c , g ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P <0.01 and ** P <0.05 (Fisher's exact test). Compound doses reflect concentrations in the food. Uncropped gels with molecular markers for d and f can be found in .

Article Snippet: Primary antibodies were as follows: rabbit anti Drosophila phospho-AKT (p-Ser505; Drosophila equivalent of mammalian AKT p-Ser473, 1:1,000, Cell Signaling, catalogue number 4054S), rabbit anti-mouse AKT (1:1,000, Cell Signaling, catalogue number 4691S), rabbit anti-mouse phospho-4EBP (Thr37/46, 1:1,000, Cell Signaling, catalogue number 2855S), rabbit anti-human phospho-AKT (Ser473, 1:1,000, Cell Signaling, catalogue number 4060S), mouse anti-chicken α-actin (1:1,000, DSHB catalogue number JLA20-s), rabbit anti-GFP (1:2,000, Sigma, catalogue number G1544), rabbit anti Drosophila P53 (1:1,000, DSHB, catalogue number H3), rabbit anti Drosophila Pten (1:1,000, gift from A. Wodarz ), mouse anti-dpERK (Thr183/Tyr185, 1:2,000, Sigma, catalogue number M8159), rabbit anti human phospho-S6 (Ser 235/236, 1:1,000, Cell Signaling, catalogue number 2211) and mouse anti Drosophila Syntaxin (1:1,000, DSHB, catalogue number 8C3).

Techniques: Western Blot, Control, Activation Assay

Journal: Nature Communications

Article Title: Functional exploration of colorectal cancer genomes using Drosophila

doi: 10.1038/ncomms13615

Figure Lengend Snippet: ( a ) Western blot analysis of PI3K signalling pathway output in ras G12V and ras G12V p53 Ri pten Ri apc Ri hindguts after 1 day feeding of SC79 at indicated doses. Syn, Syntaxin (loading control);ten hindguts per replicate. ( b ) Quantification of dissemination in ras G12V and ras G12V p53 Ri pten Ri apc Ri animals after sequential treatment with BEZ235 and indicated doses of SC79. ( c ) Quantification of dissemination in ras G12V and ras G12V p53 Ri pten Ri apc Ri animals after two different treatment schedules of SC79/BEZ235 and each drug alone. ( b , c ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P <0.01 and ** P <0.05 (Fisher's exact test). ( b ) *Variable response; not all replicates show significant rescue. Drug doses reflect concentrations in the food. Uncropped gels used to generate panel a can be found in .

Article Snippet: Primary antibodies were as follows: rabbit anti Drosophila phospho-AKT (p-Ser505; Drosophila equivalent of mammalian AKT p-Ser473, 1:1,000, Cell Signaling, catalogue number 4054S), rabbit anti-mouse AKT (1:1,000, Cell Signaling, catalogue number 4691S), rabbit anti-mouse phospho-4EBP (Thr37/46, 1:1,000, Cell Signaling, catalogue number 2855S), rabbit anti-human phospho-AKT (Ser473, 1:1,000, Cell Signaling, catalogue number 4060S), mouse anti-chicken α-actin (1:1,000, DSHB catalogue number JLA20-s), rabbit anti-GFP (1:2,000, Sigma, catalogue number G1544), rabbit anti Drosophila P53 (1:1,000, DSHB, catalogue number H3), rabbit anti Drosophila Pten (1:1,000, gift from A. Wodarz ), mouse anti-dpERK (Thr183/Tyr185, 1:2,000, Sigma, catalogue number M8159), rabbit anti human phospho-S6 (Ser 235/236, 1:1,000, Cell Signaling, catalogue number 2211) and mouse anti Drosophila Syntaxin (1:1,000, DSHB, catalogue number 8C3).

Techniques: Western Blot, Control

Journal: Frontiers in oncology

Article Title: Hypoxia-Induced LncRNA-MIR210HG Promotes Cancer Progression By Inhibiting HIF-1α Degradation in Ovarian Cancer.

doi: 10.3389/fonc.2021.701488

Figure Lengend Snippet: FIGURE 1 | Hypoxia induced lncRNA-MIR210HG in ovarian cancer. (A) qPCR analysis of lncRNA-MIR210HG expression in A2780 and SKOV3 cells treated with normoxic and hypoxia condition for 48 hours (n = 3, **p < 0.01). (B) qPCR analysis of lncRNA-MIR210HG expression in A2780 and SKOV3 cells treated with DMSO or DMOG for 48 hours (n = 3, **p < 0.01). (C) Western blotting detection of HIF-1a expression in A2780 and SKOV3 cells transfected with siRNA targeting HIF-1a and negative control under hypoxia condition. GAPDH was used as a loading control. (D) qPCR analysis of lncRNA-MIR210HG expression in A2780 and SKOV3 cells transfected with siRNA targeting HIF-1a and negative control under hypoxia condition (n = 3, **p < 0.01). (E) Detection of lncRNA-MIR210HG, U6 and 18S in A2780 and SKOV3 cells by FISH staining under hypoxia condition. Cell nucleus was stained by DAPI. Scale bar = 10 mm. (F) qPCR analysis of lncRNA-MIR210HG, GAPDH, NEAT1 and b-actin expression in nucleus and cytosol of A2780 and SKOV3 cells under hypoxia condition.

Article Snippet: After antigen retrieval in a high temperature and high pressure condition for 3 minutes, the slides were blocked with non-immune goat serum and incubated with primary antibodies against CD31 (Catalog number: 28083-1-AP, November 2021 | Volume 11 | Article 701488 Proteintech, Wuhan, China, 1:400), HIF-1a (Catalog number: 36169, CST, MA, USA, 1:100) and VEGF (Catalog number: ab46154, Abcam, London, UK, 1:100) at 4°C for overnight.

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

Journal: Frontiers in oncology

Article Title: Hypoxia-Induced LncRNA-MIR210HG Promotes Cancer Progression By Inhibiting HIF-1α Degradation in Ovarian Cancer.

doi: 10.3389/fonc.2021.701488

Figure Lengend Snippet: FIGURE 4 | lncRNA-MIR210HG inhibits HIF-1a degradation in ovarian cancer. (A) A2780 cells that were cultured under hypoxia condition were collected for RNA pull down experiments with CHIRP probe targeting lncRNA-MIR210HG and negative control. qPCR analysis of lncRNA-MIR210HG expression in A2780 cells (Input) and products of RNA pull down (n = 3, **p < 0.01). (B) Detection of HIF-1a expression in A2780 cells (Input) and products of RNA pull down. (C) Detection of HIF-1a expression in A2780 and SKOV3 cells (under hypoxia condition) stably infected with lentivirus-shRNA targeting lncRNA-MIR210HG and negative control by western blotting. GAPDH was used as a loading control. (D) Detection of HIF-1a expression in A2780-shNC and A2780-shMIR210HG cells (under hypoxia condition) treated with DMSO or MG132 by western blotting. GAPDH was used as a loading control. (E) Detection of VHL expression in A2780-shNC, A2780-shMIR210HG, SKOV3- shNC and SKOV3-shMIR210HG cells (under hypoxia condition) by western blotting. GAPDH was used as a loading control. (F) Detection of VHL expression in A2780 cells (under hypoxia condition) transfected with siRNA targeting VHL and negative control by western blotting. GAPDH was used as a loading control. (G) Detection of HIF-1a expression in A2780-shNC and A2780-shMIR210HG cells (under hypoxia condition) transfected with siRNA targeting VHL and negative control. GAPDH was used as a loading control.

Article Snippet: After antigen retrieval in a high temperature and high pressure condition for 3 minutes, the slides were blocked with non-immune goat serum and incubated with primary antibodies against CD31 (Catalog number: 28083-1-AP, November 2021 | Volume 11 | Article 701488 Proteintech, Wuhan, China, 1:400), HIF-1a (Catalog number: 36169, CST, MA, USA, 1:100) and VEGF (Catalog number: ab46154, Abcam, London, UK, 1:100) at 4°C for overnight.

Techniques: Cell Culture, Negative Control, Expressing, Stable Transfection, Infection, shRNA, Western Blot, Control, Transfection

Journal: Frontiers in oncology

Article Title: Hypoxia-Induced LncRNA-MIR210HG Promotes Cancer Progression By Inhibiting HIF-1α Degradation in Ovarian Cancer.

doi: 10.3389/fonc.2021.701488

Figure Lengend Snippet: FIGURE 5 | Knockdown of lncRNA-MIR210HG inhibits tumor growth in mice. (A) A2780-shNC and A2780-shMIR210HG cells were injected into nude mice to establish subcutaneous tumor model. Tumor volume was measured every 5 days and the tumor growth curve was performed (n = 5, **p < 0.01). (B) Tumor weight of A2780-shNC and A2780-shMIR210HG tumors (n = 5, **p < 0.01). (C) qPCR analysis of lncRNA-MIR210HG expression in A2780-shNC and A2780-shMIR210HG tumors (n = 3, **p < 0.01). (D) SKOV3-shNC and SKOV3-shMIR210HG cells were injected into nude mice to establish subcutaneous tumor model. Tumor volume was measured every 5 days and the tumor growth curve was performed (n = 5, **p < 0.01). (E) Tumor weight of SKOV3-shNC and SKOV3-shMIR210HG tumors (n = 5, **p < 0.01). (F) qPCR analysis of lncRNA-MIR210HG expression in SKOV3-shNC and SKOV3-shMIR210HG tumors (n = 3, **p < 0.01). (G) Detection of tumor angiogenesis in A2780-shNC and A2780-shMIR210HG tumors by CD31 staining. Cell nucleus was stained with DAPI. The number of tumor vessels was analyzed (n = 3, **p < 0.01, Scale bar = 100 mm). (H) Detection of HIF-1a in A2780-shNC and A2780-shMIR210HG tumors by immunofluorescent staining. Cell nucleus was stained with DAPI. The percent of HIF-1a positive cells was analyzed (n = 3, **p < 0.01, Scale bar = 100 mm). (I) Detection of VEGF in A2780-shNC and A2780-shMIR210HG tumors by IHC staining. The percent of VEGF positive cells was analyzed (n = 3, **p < 0.01, Scale bar = 100 mm).

Article Snippet: After antigen retrieval in a high temperature and high pressure condition for 3 minutes, the slides were blocked with non-immune goat serum and incubated with primary antibodies against CD31 (Catalog number: 28083-1-AP, November 2021 | Volume 11 | Article 701488 Proteintech, Wuhan, China, 1:400), HIF-1a (Catalog number: 36169, CST, MA, USA, 1:100) and VEGF (Catalog number: ab46154, Abcam, London, UK, 1:100) at 4°C for overnight.

Techniques: Knockdown, Injection, Expressing, Staining, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Molecular interactions of adaptor protein PSTPIP2 control neutrophil-mediated responses leading to autoinflammation

doi: 10.3389/fimmu.2022.1035226

Figure Lengend Snippet: Neutrophil recruitment to the sites of inflammation. (A) Detection of neutrophil elastase and Ly6G in the hind paw lysates as a hallmark of neutrophil presence. (B) Percentages of activated (CD62L - ) neutrophils within total neutrophils isolated from hind paws of the mice of indicated mouse strains measured by flow cytometry. (C-F) Concentrations of IL-17A/F (C) , CCL3 (D) , CXCL1 (E) , and CXCL2 (F) in hind paw lysates detected by ELISA. (G) Cxcl2 mRNA levels in neutrophils purified from hind paws of the mice of indicated mouse strains determined by quantitative RT-PCR. Error bars represent mean ± SEM. Asterisks above individual columns describe p values for comparisons with WT, asterisks above connecting lines describe p values for comparisons of the columns connected by these lines; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. See Materials and Methods for further details on statistical analysis.

Article Snippet: ELISA was performed according to manufacturer’s instructions using IL-1 beta Mouse Uncoated ELISA Kit, MIP-2/CXCL2 Mouse ELISA Kit, MIP-1a (CCL3) Mouse Uncoated ELISA Kit (Invitrogen, ThermoFisher Scientific, catalog numbers 88-7013-88, EMCXCL2, and 88-56013-88), Mouse IL-17A/F Heterodimer DuoSet ELISA, and Mouse CXCL1/KC DuoSet ELISA DY5390-05, and DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems, catalog numbers DY5390-05, DY453-05, DY008).

Techniques: Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Purification, Quantitative RT-PCR

Journal: Biology of Sex Differences

Article Title: Targeting senescence improves angiogenic potential of adipose-derived mesenchymal stem cells in patients with preeclampsia

doi: 10.1186/s13293-019-0263-5

Figure Lengend Snippet: Senescent cell burden is higher in PE-MSC compared to NP-MSC. SABG staining revealed a higher number of stained cells (marked with black arrows) in PE-MSC compared to their normotensive counterparts ( a ). Data presented as mean values of SABG-stained MSC with min-max ( b ). Expression of p16, but not p21, was significantly increased. All SASP genes were significantly more highly expressed in PE-MSC compared to NP-MSC. Data are shown as box-plots (min-max) with all individual values ( c ). PE-MSC and NP-MSC were co-cultured with GFP expressing HUVEC for 8 days in total, and total network length was measured continuously every 3 h. Significantly lower angiogenic potential was registered for HUVEC co-cultured with PE-MSC, compared to NP-MSC ( F = 13.965; df = 8, p < 0.001) ( d )

Article Snippet: The following primers were purchased from Applied Biosciences: total p16 (catalog number: Hs00923894), p21 (catalog number: Hs00355782), IL-6 (catalog number: Hs00174131), IL-8 (catalog number: Hs00174103), MCP-1 (catalog number: Hs00234140), PAI-1 (catalog number: Hs01126607), and PAI-2 (catalog number: Hs00299953).

Techniques: Staining, Expressing, Cell Culture

Journal: Biology of Sex Differences

Article Title: Targeting senescence improves angiogenic potential of adipose-derived mesenchymal stem cells in patients with preeclampsia

doi: 10.1186/s13293-019-0263-5

Figure Lengend Snippet: Treatment with dasatinib cleared senescent cells from PE-MSC and affected senescence-related gene expression. Representative images from SABG staining show abundant senescent cells in PE-MSC (marked with black arrows), but not in NP-MSC, in vehicle ( a ). Dasatinib treatment completely removed senescent cells from PE-MSC ( p < 0.001) ( b ). PE-MSC had a significant decrease in expression of p16 ( p = 0.025) and PAI-1 ( p < 0.001), IL-6 ( p = 0. 0487), and MCP-1 ( p = 0.040), while IL-8 ( p = 0.136) was modestly decreased after treatment. Significantly increased expression of the p21 and PAI-2 genes was observed after the treatment ( c ). Relative expression of the senescence marker gene, p16, in NP-MSC after treatment with Dasatinib remained unchanged ( p = 0.136). Apart from p21 and PAI-2 whose expression increased in a manner similar to PE-MSC, the relative gene expression of the other tested genes was decreased in NP-MSC after the treatment with dasatinib ( p < 0.001) ( d )

Article Snippet: The following primers were purchased from Applied Biosciences: total p16 (catalog number: Hs00923894), p21 (catalog number: Hs00355782), IL-6 (catalog number: Hs00174131), IL-8 (catalog number: Hs00174103), MCP-1 (catalog number: Hs00234140), PAI-1 (catalog number: Hs01126607), and PAI-2 (catalog number: Hs00299953).

Techniques: Gene Expression, Staining, Expressing, Marker

Journal: Frontiers in Medicine

Article Title: Exposure to Plasma From Non-alcoholic Fatty Liver Disease Patients Affects Hepatocyte Viability, Generates Mitochondrial Dysfunction, and Modulates Pathways Involved in Fat Accumulation and Inflammation

doi: 10.3389/fmed.2021.693997

Figure Lengend Snippet: Effects of NAFLD plasma on PPARγ (A) and SREBP-1c (B) expression in Huh7.5 cells. P, patient; M, MCC950 (1 nM, for 30 min). Other abbreviations are as described in previous Figures. In the densitometric analysis, all values are normalized vs. control which is normalized, as well, and considered as 1. They are shown as fold changes vs. control. Reported data are means ± SD of five independent experiments for each experimental protocol.

Article Snippet: After electrophoresis they were transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories) and incubated overnight at 4°C with specific primary antibodies: anti Nf-kB (p-50; 1:1,000; Santa Cruz Biotechnology, catalog number sc-8414), anti gp91-phox (NOX2; 1:1,000; Santa Cruz Biotechnology, catalog number sc-130543), anti PPAR-γ (1:1,000; Santa Cruz Biotechnology, catalog number sc-271392), anti SREBP-1c (1:1,000; Santa Cruz Biotechnology, catalog number sc-13551).

Techniques: Clinical Proteomics, Expressing, Control