Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 1 The expression of DEF6 is increased in hypertrophic hearts and cardiomyocytes. A mRNA levels of DEF6 in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 5). B Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 4). C mRNA levels of DEF6 in NRCMs administrated with PBS or 24 h of PE (50 μM) (n = 5). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in NRCMs administrated with PBS or 24 h of PE (n = 4). *P < 0.05, ***P < 0.001 vs. sham or PBS. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Student’s t test (A, C) or Mann–Whitney U test (B, D).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 2 Ablation of DEF6 mitigates TAC-induced cardiac hypertrophy. A Strategy to construct KO mice and the sequencing results of WT and KO mice. B Protein levels of cardiac DEF6 in WT and KO mice (n = 5). C Comparisons of HW, HW/BW, LW/BW, and HW/TL in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). D Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area from groups (n = 6). E RT-PCR analyses of the hypertrophic markers in the indicated groups (n = 4). F–H Comparisons of the LVEDd, LVESd, LVPWd, FS, and EF values in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). I Left, PSR-stained LV sections in WT and KO mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). J RT-PCR analysis of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by One-way ANOVA (C, D, F–I) or Kruskal–Wallis test (E, J).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Construct, Sequencing, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 3 Overexpression of DEF6 aggravates TAC-induced cardiac hypertrophy. A mmunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the hearts of mice injected with AAV9-vector or AAV9-DEF6 (n = 4). B Comparisons of HW, HW/BW, LW/ BW, and HW/TL in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). C Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area between groups (n = 6). D RT-PCR analyses of the hypertrophic markers in each groups (n = 4). E–G Comparisons of LVEDd, LVESd, LVPWd, FS, and EF in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). H Left, PSR-stained LV sections in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). I RT-PCR analyses of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AAV9- vector or AAV9-vector sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AAV9-vector TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by Mann–Whitney U test (A) or One-way ANOVA (B, C, E–H) or Kruskal–Wallis test (D, I).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Over Expression, Expressing, Injection, Plasmid Preparation, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 4 DEF6 exacerbates PE-induced cardiomyocyte hypertrophy. A Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCMs infected with AdshRNA or AdshDEF6 (n = 3). B Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with AdshRNA or AdshDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). C RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCVs infected with Advector or AdDEF6 (n = 3). E Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with Advector or AdDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). F RT-PCR analyses of the hypertrophic markers in cultured NRCVs of each groups (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AdshRNA or AdshRNA PBS or Advector or Advector PBS, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AdshRNA PE or Advector PE. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Mann–Whitney U test (A, D) or One-way ANOVA (B, E) or Kruskal–Wallis test (C, F).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Western Blot, Expressing, Cell Culture, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 6 Prohypertrophic effect of DEF6 depends on Rac1-MEK-ERK signaling. A Co-IP of DEF6 was performed with anti-Flag and probed by Western blots with anti-HA (left); Co-IP of Rac1 was performed with anti-HA and probed by Western blots with anti-Flag (right). B In vitro GST pulldown assays for the interaction of purified Flag-DEF6 and GST-HA-Rac1 (left), as well as Flag-Rac1 and GST-HA-DEF6 (right). C The activity of Rac1 changes in the same direction as the DEF6 expression. D Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with Advector or AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM) (n = 3). E Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with Advector and AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM). (n ≥48 cells per group). F RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). G Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with AdshRNA or AdshDEF6 and with Adcontrol or AdRac1(G12V) under PE 24 h of PE treatment (50 μM) (n = 3). H Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with the indicated adenovirus and administrated with 24 h of PE (50 μM) (n ≥48 cells per group). I RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). •P < 0.05, P < 0.01,•P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, ###P < 0.001 vs. AdDEF6 PBS PE or AdshDEF6 Adcontrol PE, and n.s. indicates no significance. Data are displayed as mean ± SD. Statistical analysis were conducted by Kruskal–Wallis test.

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Activity Assay, Expressing, Cell Culture, Infection, Staining, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Myosin VI Reduces Proliferation, but Not Differentiation, in Pluripotent P19 Cells

doi: 10.1371/journal.pone.0063947

Figure Lengend Snippet: (A) Mouse embryonal P19 cells were stably transfected with Myo6 expression vector, followed by selection by G418 and subsequent determination of Myo6 mRNA expression with RT-PCR analysis. Several clones of stable transfectants were cultured with ATRA for 4 days for subsequent (B) micrographic observation and (C) MTT reduction determination. Each value represents the mean ± S.E. in 4 different experiments. *P<0.05, **P<0.01, significantly different from the control value in stable EV transfectants.

Article Snippet: After 24 h, and every 48 h thereafter for 2 weeks, the culture medium was replaced with fresh medium containing 500 μg/ml of G418 (Invitrogen).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Selection, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Cell Culture, Control

Journal: The Journal of Biological Chemistry

Article Title: Identification of Ror2 as a Hypoxia-inducible Factor Target in von Hippel-Lindau-associated Renal Cell Carcinoma *

doi: 10.1074/jbc.M109.073924

Figure Lengend Snippet: Minimal hypoxia induction of Ror2 expression. A, Ror2 expression is minimally induced after 48 h of exposure to cobalt chloride. Normoxic (N) and CoCl2 (C)-treated protein samples were immunoblotted with HIF-2α and Egln3 antibodies to show that HIF-2α and the HIF target Egln3 were induced in VHL(+) cell lines. Ror2 levels remained stable to this manipulation. Ku80 antibody was used as a loading control (LC). B, left, ROR2 mRNA levels are minimally induced under hypoxic-like conditions in RCC4 cells. qRT-PCR analysis of the hypoxia mimetic cobalt chloride (CoCl2) transcriptional induction after 24 h in the VHL expressing cell line RCC4 3-14 demonstrate induction of the HIF target gene EGLN3 (**, p = 0.0015) in response to treatment with hypoxia mimetic, confirming HIF transcriptional activity. Ror2 mRNA was not significantly induced under these conditions. Right, Ror2 mRNA levels are minimally induced under hypoxic-like conditions in 786-0 WT8 cells. qRT-PCR analysis of the hypoxia mimetic DMOG transcriptional induction after 24 h in the VHL expressing cell line 786-0 WT8 demonstrate induction of HIF target genes EGLN3 (**, p = 0.0091) and GLUT1 (**, p = 0.0015) in response to treatment with the hypoxia mimetic, confirming HIF transcriptional activity. Ror2 transcript levels show minimal enrichment upon treatment. Transcript values are shown as normalized to the β-actin RNA internal standard and relative to the unstimulated cells of each set of paired cells. Error bars represent ± S.E.

Article Snippet: cDNA was made by reverse transcription from 0.5 μg of total RNA (5PRIME Perfect Pure RNA Cultured Cell Kit, Gaithersburg, MD) using oligo(dT) primers and the SuperScript II RT-PCR reagents (Invitrogen).

Techniques: Expressing, Control, Quantitative RT-PCR, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of Ror2 as a Hypoxia-inducible Factor Target in von Hippel-Lindau-associated Renal Cell Carcinoma *

doi: 10.1074/jbc.M109.073924

Figure Lengend Snippet: Ror2 is regulated by VHL status. A, Ror2 is expressed when VHL is mutated in the 786-0 paired cell line. Whole cell extracts from 786-0 cells expressing (786-0 WT8) or lacking expression (786-0 RC3) of pVHL were immunoblotted with monoclonal Ror2 antibody (Ror2(1)), polyclonal Ror2 antibody (Ror2(2)), and Ku80 antibody as a loading control (LC). B, Ror2 expression is regulated at the transcriptional level in the paired 786-0 cell lines. qRT-PCR analysis data are normalized to β-actin as an RNA internal standard and displayed as expression levels relative to the pVHL expressing member of the cell line pair. Significant differences were observed as ROR2 mRNA levels were suppressed in the VHL(+) cell line (light gray), relative to the paired vector-transfected VHL(−) control (dark gray), **, p < 0.0001. C, Ror2 is expressed when VHL is mutated in the RCC4 paired cell line. Whole cell extracts from RCC4 cells expressing (RCC4 3-14) or lacking expression (RCC4 2-1) of pVHL were immunoblotted with monoclonal Ror2 antibody (Ror2(1)), polyclonal Ror2 antibody (Ror2(2)), and Ku80 antibody as a LC. D, Ror2 expression is regulated at the transcriptional level in the paired RCC4 cell lines. qRT-PCR analysis data are normalized to β-actin as an RNA internal standard and displayed as expression levels relative to the pVHL expressing member of the cell line pair. Significant differences observed as ROR2 mRNA levels were suppressed in the VHL(+) cell line (light gray), relative to the paired vector-transfected VHL(−) control (dark gray), **, p = 0.0001. Error bars represent mean ± S.E.

Article Snippet: cDNA was made by reverse transcription from 0.5 μg of total RNA (5PRIME Perfect Pure RNA Cultured Cell Kit, Gaithersburg, MD) using oligo(dT) primers and the SuperScript II RT-PCR reagents (Invitrogen).

Techniques: Expressing, Control, Quantitative RT-PCR, Plasmid Preparation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Identification of Ror2 as a Hypoxia-inducible Factor Target in von Hippel-Lindau-associated Renal Cell Carcinoma *

doi: 10.1074/jbc.M109.073924

Figure Lengend Snippet: Ror2 is regulated by HIF-2α expression. A, Ror2 expression is suppressed in 786-0 HIF-2α shRNA knockdown cells. RCC cells expressing a VHL transgene (786-0 WT8) were used to control for VHL-induced suppression of Ror2. Cells lacking expression of VHL (786-0) were further transfected with a control scramble shRNA (scramble) or a HIF-2α short hairpin RNA retrovirus (shHIF-2) and immunoblotted with HIF-2α, Ror2, and Ku80 (loading control, LC) antibodies. B, ROR2 mRNA levels are suppressed in 786-0 HIF-2α shRNA knockdown cells. qRT-PCR analysis of the 786-0 HIF-2α shRNA knockdown cells verified effective HIF-2α knockdown in two independent knockdown cell lines (**, p = 0.0022, p = 0.0001). Ror2 expression was concordantly suppressed with HIF-2α knockdown (*, p = 0.0172; **, p = 0.0023). C, Ror2 expression can be rescued by HIF-2α overexpression. 786-0 cells stably expressing scramble shRNA and shHIF-2 were rescued with transient expression of empty vector (pcDNA) or proline hydroxylation-resistant double proline to alanine HIF-2α mutant (DPA) for 48 and 72 h. Induction of Ror2 expression is detected when HIF-2α expression is rescued with the DPA HIF-2α mutant in the shRNA cell line. Error bars represent ± S.E.

Article Snippet: cDNA was made by reverse transcription from 0.5 μg of total RNA (5PRIME Perfect Pure RNA Cultured Cell Kit, Gaithersburg, MD) using oligo(dT) primers and the SuperScript II RT-PCR reagents (Invitrogen).

Techniques: Expressing, shRNA, Knockdown, Control, Transfection, Quantitative RT-PCR, Over Expression, Stable Transfection, Plasmid Preparation, Mutagenesis

Journal: Frontiers in Plant Science

Article Title: Combined metabolomic and transcriptomic analysis reveals key components of OsCIPK17 overexpression improves drought tolerance in rice

doi: 10.3389/fpls.2022.1043757

Figure Lengend Snippet: PC biplot of the transcriptome and metabolome of rice seedlings under drought stress and identification of differentially expressed genes and metabolites. (A) PC biplot of transcriptome. (B) PC biplot of metabolome. (C) Volcano plot of differentially expressed genes between NIP and OsCIPK17 -OE9 under drought treatment. (D) Volcano plot of differentially expressed metabolites between NIP and OsCIPK17 -OE9 under drought treatment. (E) KEGG analysis of differentially expressed genes and metabolites between NIP and OsCIPK17 -OE9 under drought treatment. CK means control check, which means growth under normal conditions without drought treatment. D means under drought treatment. OE9 means OsCIPK17 overexpression line 9. NIP-D, NIP-CK, OE9-D, and OE9-CK mean combination of lines and treatment methods. The same meaning as the following figure.

Article Snippet: Suzhou PANOMIX Biomedical Tech Co., LTD sequenced the transcriptome of NIP and OE9 roots under normal (NIP-CK and OE9-CK) and drought (NIP-D and OE9-D) conditions.

Techniques: Control, Over Expression

Journal: Frontiers in Plant Science

Article Title: Combined metabolomic and transcriptomic analysis reveals key components of OsCIPK17 overexpression improves drought tolerance in rice

doi: 10.3389/fpls.2022.1043757

Figure Lengend Snippet: Metabolites and enzymes related to trehalose metabolism. (A) Contents of metabolites in the trehalose pathway. Black boxes indicate the substances detected by the metabolomic analysis, and red boxes indicate the substances not detected by the metabolomic analysis. (B) Expression of enzymes involved in trehalose pathway detected in the transcriptome. Data are presented as mean ± SD of six independent experiments, and significant differences between NIP-CK and OE9-CK/NIP-D or OE9-D and OE9-CK/NIP-D plants according to one-way ANOVA are indicated by asterisks. CK: NIP or OE9 materials without drought treatment. OE9: OsCIPK17 -OE9. D: drought treatment. Asterisks indicate significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Suzhou PANOMIX Biomedical Tech Co., LTD sequenced the transcriptome of NIP and OE9 roots under normal (NIP-CK and OE9-CK) and drought (NIP-D and OE9-D) conditions.

Techniques: Expressing