Journal: mSystems

Article Title: Nuclear Ssr4 Is Required for the In Vitro and In Vivo Asexual Cycles and Global Gene Activity of Beauveria bassiana

doi: 10.1128/mSystems.00677-19

Figure Lengend Snippet: Properties of Ssr4 in B. bassiana . (A) Structural comparison of Ssr4 orthologs found in B. bassiana (Bba), Cordyceps militaris (Cmi), Fusarium fugikuroi (Ffu), Metarhizium roberstii (Mro), Trichoderma reesei (Tre), Aspergillus nidulans (Ani), Penicillium digitatum (Pdi), and S. pombe (Spo). The main domains and nuclear localization signal (NLS) were predicted at http://smart.embl-heidelberg.de and http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi , respectively. (B) Relative transcript (RT) level of ssr4 in the SDAY culture of a WT strain during a 7-day incubation at the optimal regimen with respect to a standard at the end of 48 h. Values are means ± standard deviations (SD) (error bars) from three cDNA samples. Different lowercase letters denote significant differences (Tukey’s HSD test, P < 0.05). (C) LSCM images for subcellular localization of GFP::Ssr4 fusion protein in the hyphal cells collected from 3-day-old SDBY culture and stained with DAPI. Panels 1 to 4 show bright, expressed, stained, and merged views of the same field, respectively. Note that the expressed signal (green) overlapped well with the stained signal (blue) in nuclei. Bars, 5 μm. (D) Western blots for GFP (27 kDa) and GFP::Ssr4 (102.9 kDa) in the protein extracts isolated from the 3-day-old SDBY cultures of transgenic strain (TS) versus WT (control) and probed with anti-GFP antibodies.

Article Snippet: The protein extracts isolated from the 3-day-old cultures of the WT and transgenic strains were probed with mouse monoclonal anti-GFP antibodies (Cell Signaling Technology, Boston, MA, USA) for Western blots of GFP alone and GFP::Ssr4 to verify whether the fusion protein was expressed as expected.

Techniques: Incubation, Staining, Western Blot, Isolation, Transgenic Assay

Journal: Cancers

Article Title: HSF1 Stimulates Glutamine Transport by Super-Enhancer-Driven lncRNA LINC00857 in Colorectal Cancer

doi: 10.3390/cancers14163855

Figure Lengend Snippet: The HSF1/LINC00857 axis promotes the transcription of ANXA11. ( A ) Subcellular localization of LINC00857 in HCT116 and DLD1 by FISH (scale bar, 10 µm). ( B ) Diagram of LINC00857 promoter regions. The scatter plot of the relationship between HSF1, LINC00857, and ANXA11. ( C ) Correlation between LINC00857 and ANXA11 expression using the GEPIA database. ( D ) HSF1 and ANXA11 expression levels as detected in the normal and tumor tissues by IHC (immunohistochemistry) (scale bar, 50 µm and 100 µm). ( E ) Relative ANXA11 mRNA expression after LINC00857 knockdown. ( F ) Relative ANXA11 mRNA expression after HSF1 knockdown. ( G ) Relative luciferase activity after transfection HSF1, LINC00857, and ANXA11 promoter. ( H ) Relative enrichment level of RNA Pol II at the ANXA11 promoter after LINC00857 or HSF1 knockdown by ChIP–qPCR assays. All data represent the mean ± SD, n = 3. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The following antibodies were used in this study: a-Tubulin (T5168, Sigma-Aldrich, St. Louis, MO, USA), HSF1 (ab52757, Abcam, Cambridge, UK), Histone H3 (acetyl K27) (ab177178, Abcam), ANXA11 (SC46686, Santa Cruz, CA, USA), p-mTOR (ab109268, Abcam), p-P70S6K (9234S, CST, Danvers, MA, USA), SLC1A5 (ab237704, Abcam), P300 (ab275378, Abcam), Pol II (ab26721, Abcam), BRD4 (A301-985A100, Bethyl Laboratories, Montgomery, TX, USA), β-actin (4970L, CST).

Techniques: Expressing, Immunohistochemistry, Luciferase, Activity Assay, Transfection

Journal: Cancers

Article Title: HSF1 Stimulates Glutamine Transport by Super-Enhancer-Driven lncRNA LINC00857 in Colorectal Cancer

doi: 10.3390/cancers14163855

Figure Lengend Snippet: The LINC00857/ANXA11 axis promotes SLC1A5-mediated glutamine transport. ( A ) Score plot of principal components analysis (PCA) between control and LINC00857 knockdown group after LC–MS (liquid chromatography mass spectrometry) analysis. ( B ) Heatmap of 22 amino acids’ alteration after LINC00857 knockdown. ( C ) Association analysis between different amino acid. ( D ) Concentration of Glu (glutamic acid) and Asp (aspartic acid) after silencing LINC00857. ( E ) Correlation between ANXA11 and SLC1A5 expression using the GEPIA database. ( F ) The effect of ANXA11 knockdown on SLC1A5 expression in CRC cells, as explored by Western blotting. ( G , H ) Bioinformatics tools predicted a series of miRNAs complementary to ANXA11 mRNA and the SLC1A5 3′UTR region. ( I ) Dual-luciferase assays of the ANXA11 mRNA/SLC1A5 3′UTR constructions with intact or mutated seed sequences for miR-122-5p. ( J ) The effect of miR-122-5p mimics on SLC1A5 protein level, as explored by western blotting. ( K ) The effect of miR-122-5p inhibitors on ANXA11 knockdown-induced SLC1A5 downregulation, as explored by Western blotting. ( L , M ) RIP (RNA immunoprecipitation) assays were performed using the Ago2 antibody. The relative expression levels of ANXA11 and miR-122-5p were determined by qPCR. All data represent the mean ± SD, n = 3. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The following antibodies were used in this study: a-Tubulin (T5168, Sigma-Aldrich, St. Louis, MO, USA), HSF1 (ab52757, Abcam, Cambridge, UK), Histone H3 (acetyl K27) (ab177178, Abcam), ANXA11 (SC46686, Santa Cruz, CA, USA), p-mTOR (ab109268, Abcam), p-P70S6K (9234S, CST, Danvers, MA, USA), SLC1A5 (ab237704, Abcam), P300 (ab275378, Abcam), Pol II (ab26721, Abcam), BRD4 (A301-985A100, Bethyl Laboratories, Montgomery, TX, USA), β-actin (4970L, CST).

Techniques: Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Concentration Assay, Expressing, Western Blot, Luciferase, Immunoprecipitation

Journal: Cancers

Article Title: HSF1 Stimulates Glutamine Transport by Super-Enhancer-Driven lncRNA LINC00857 in Colorectal Cancer

doi: 10.3390/cancers14163855

Figure Lengend Snippet: Knockout of ANXA11 attenuated CRC carcinogenesis in vivo. ( A ) Expression of ANXA11 protein in azoxymethane (AOM)/dextran sulfate sodium (DSS)–treated mice compared with normal mice. ( B ) Expression of ANXA11 protein in AOM/DSS–treated mice after HSF1 knockout. ( C ) Schematic digraphs depicting the CRISPR/Cas9-mediated ANXA11 knockout strategy. ( D ) Representative images of ANXA11 +/+ and ANXA11 −/− mice colon and rectum. ( E ) Assessment of the number of tumors of ANXA11 +/+ and ANXA11 −/− mice. ( F ) Assessment of the tumor area of ANXA11 +/+ and ANXA11 −/− mice. ( G ) The overall survival of ANXA11 +/+ and ANXA11 −/− mice. ( H ) The body weight of ANXA11 +/+ and ANXA11 −/− mice. ( I ) Representative HE (hematoxylin–eosin) staining and IHC (immunohistochemistry) staining of Ki67 and ANXA11 from the ANXA11 +/+ and ANXA11 −/− mice colorectal tissues (scale bar, 20 µm). ( J ) Working model. All data represent the mean ± SD, n ≥ 3. **** p < 0.0001.

Article Snippet: The following antibodies were used in this study: a-Tubulin (T5168, Sigma-Aldrich, St. Louis, MO, USA), HSF1 (ab52757, Abcam, Cambridge, UK), Histone H3 (acetyl K27) (ab177178, Abcam), ANXA11 (SC46686, Santa Cruz, CA, USA), p-mTOR (ab109268, Abcam), p-P70S6K (9234S, CST, Danvers, MA, USA), SLC1A5 (ab237704, Abcam), P300 (ab275378, Abcam), Pol II (ab26721, Abcam), BRD4 (A301-985A100, Bethyl Laboratories, Montgomery, TX, USA), β-actin (4970L, CST).

Techniques: Knock-Out, In Vivo, Expressing, CRISPR, Staining, Immunohistochemistry