Journal: eLife

Article Title: PHLPP1 counter-regulates STAT1-mediated inflammatory signaling

doi: 10.7554/eLife.48609

Figure Lengend Snippet: ( A ) Luciferase reporter assay in HEK-293T cells over-expressing GAS luciferase reporter in combination with either vector control (Vector, black), PHLPP1 NTE (NTE, green), PHLPP1ΔNTE (blue), or PHLPP1 (red) and treated with 10 ng/ml IFNγ for 0, 1, 6, or 24 hr. Values are expressed as mean of RLU ± SEM of five independent experiments. All data points at 24 hr were significant against each other except for vector to PHLPP1ΔNTE, and PHLPP1ΔNTE to PHLPP1. *p<0.05, **p<0.01, ***p<0.001 (Student’s t -test). ( B ) Western blot analysis of detergent-solubilized lysate of HEK-293T cells transfected with vector control (Vector), HA-tagged NTE of PHLPP1 (HA-NTE), PHLPP1 with the NTE deleted (HA-P1ΔNTE) or full-length PHLPP1 (HA-P1) and immunoprecipitated (IP) using HA antibody; blots were probed for co-IP of STAT1 tag using GFP antibody. ( C ) Quantification of GFP-STAT1 IP divided by HA IP and normalized to HA-NTE IP. Values are expressed as mean ± SEM of three independent experiments. ***p<0.001, ****p<0.0001 (Student’s t -test).

Article Snippet: Antibody against HA (11867425001) was purchased from Roche; antibodies against GFP (2555), STAT1 (9172), phosphorylated Ser727 on STAT1 (9177), phosphorylated Tyr701 on STAT1 (7649), phosphorylated Thr202/Tyr204 on Erk1/2 (9101), total Erk1/2 (9102), and phosphorylated Thr308 on Akt (9275) were purchased from Cell Signaling.

Techniques: Luciferase, Reporter Assay, Expressing, Plasmid Preparation, Control, Western Blot, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: eLife

Article Title: PHLPP1 counter-regulates STAT1-mediated inflammatory signaling

doi: 10.7554/eLife.48609

Figure Lengend Snippet:

Article Snippet: Antibody against HA (11867425001) was purchased from Roche; antibodies against GFP (2555), STAT1 (9172), phosphorylated Ser727 on STAT1 (9177), phosphorylated Tyr701 on STAT1 (7649), phosphorylated Thr202/Tyr204 on Erk1/2 (9101), total Erk1/2 (9102), and phosphorylated Thr308 on Akt (9275) were purchased from Cell Signaling.

Techniques: Derivative Assay, Stable Transfection, Expressing, Control, Infection, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Immunofluorescence, Recombinant, Sequencing, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Characterization of the Mitochondrial Localization of the Nuclear Receptor SHP and Regulation of Its Subcellular Distribution by Interaction with Bcl2 and HNF4α

doi: 10.1371/journal.pone.0068491

Figure Lengend Snippet: (A) The subcellular localization of GFP-hSHP in 293T cells was observed under a fluorescence microscope, and HNF4α was visualized by immunofluorescence using a specific HNF4α antibody. Scale bars: 100 µm, 50 µm (insets). (B) The N-terminal domain of SHP is primarily responsible for the interaction with HNF4α protein. Left, Diagram showing the deletion mutation constructs of GFP-mShp. Right, 293T cells were transfected with GFP-mSHP deletion constructs (4 µg, 6 cm plate) along with HA-HNF4α (4 µg, 6 cm plate) expression vector. Anti-HA agarose was used to immuneprecipitate HNF4α, and the protein levels of various GFP-mSHP truncation mutants and HNF4α were detected by Western blots using anti-GFP or anti-HA antibodies, respectively. Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.

Article Snippet: The VDAC antibody (#4661), COX IV antibody (#4850), Hsp60 antibody (#4870), Cyto c antibody (#4280), PARP antibody (#9532), Bcl2 antibody (#2870), myc antibody (#2278), GFP antibody (#2955), GST antibody(#2625), and HNF4α antibody (#3113) were purchased from Cell Signaling.

Techniques: Fluorescence, Microscopy, Immunofluorescence, Mutagenesis, Construct, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Characterization of the Mitochondrial Localization of the Nuclear Receptor SHP and Regulation of Its Subcellular Distribution by Interaction with Bcl2 and HNF4α

doi: 10.1371/journal.pone.0068491

Figure Lengend Snippet: The subcellular localization of GFP-mSHP full length and deletion mutants in 293T cells was observed by fluorescence microscopy, and HNF4α was visualized by immunofluorescence using a specific HNF4α antibody. Scale bars: 50 µm. Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.

Article Snippet: The VDAC antibody (#4661), COX IV antibody (#4850), Hsp60 antibody (#4870), Cyto c antibody (#4280), PARP antibody (#9532), Bcl2 antibody (#2870), myc antibody (#2278), GFP antibody (#2955), GST antibody(#2625), and HNF4α antibody (#3113) were purchased from Cell Signaling.

Techniques: Fluorescence, Microscopy, Immunofluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: METTL16 promotes hepatocellular carcinoma progression through downregulating RAB11B-AS1 in an m 6 A-dependent manner

doi: 10.1186/s11658-022-00342-8

Figure Lengend Snippet: The expression pattern and clinical association of METTL16 in HCC. A METTL16 mRNA expression in human HCC tissues ( n = 95) and normal liver tissues ( n = 39) from GSE45436 dataset. **** P < 0.0001 by Mann–Whitney test. B METTL16 mRNA expression level in 63 pairs of HCC tissues and matched adjacent noncancerous liver tissues was measured by RT-qPCR. **** P < 0.0001 by Wilcoxon signed-rank test. C METTL16 mRNA expression level in HCC cell lines HepG2, SNU-398, Huh7, and normal hepatic cell line TLHE-2 was measured by RT-qPCR. Results are shown as mean ± standard deviation (SD) of n = 3 independent experiments. * P < 0.05 by one-way ANOVA followed by Dunnett’s multiple comparisons test. D Kaplan–Meier survival analysis of the correlation between METTL16 mRNA expression and overall survival based on TCGA liver cancer data, analyzed by the online in silico tool Kaplan–Meier Plotter ( https://kmplot.com/analysis/ ). E Kaplan–Meier survival analysis of the correlation between METTL16 mRNA expression and overall survival in our HCC cohort. n = 63 patients with HCC. P = 0.0355 by log-rank test. Median METTL16 level was used as cutoff

Article Snippet: After incubation with primary antibodies against METTL16 (78 kDa, #17676, 1:1000, Cell Signaling Technology, Danvers, MA, USA) or GAPDH (36 kDa, 60004-1-Ig, 1:5000, Proteintech, Chicago, IL, USA) at 4 °C overnight, the membranes were further incubated with IRDye 680RD Goat anti-Mouse IgG Secondary Antibody (926-68070, 1:10000, Li-Cor Biosciences, Lincoln, NE, USA) or IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, 1:10000, Li-Cor).

Techniques: Expressing, MANN-WHITNEY, Quantitative RT-PCR, Standard Deviation, In Silico

Journal: Cellular & Molecular Biology Letters

Article Title: METTL16 promotes hepatocellular carcinoma progression through downregulating RAB11B-AS1 in an m 6 A-dependent manner

doi: 10.1186/s11658-022-00342-8

Figure Lengend Snippet: The roles of METTL16 overexpression in HCC. A METTL16 protein expression in SNU-398 and HepG2 cells with METTL16 overexpression or control was detected by western blot. B Migration ability of SNU-398 and HepG2 cells with METTL16 overexpression or control was detected by transwell migration assay. Scale bars, 100 µm. C Invasion ability of SNU-398 and HepG2 cells with METTL16 overexpression or control was detected by transwell invasion assay. Scale bars, 100 µm. D Cellular proliferation of SNU-398 cells with METTL16 overexpression or control was detected by EdU assay. Scale bars, 100 µm. E Cellular proliferation of HepG2 cells with METTL16 overexpression or control was detected by EdU assay. Scale bars, 100 µm. F Cellular proliferation of SNU-398 cells with METTL16 overexpression or control was detected by CCK-8 assay. G Cellular proliferation of HepG2 cells with METTL16 overexpression or control was detected by CCK-8 assay. H Cellular apoptosis of SNU-398 cells with METTL16 overexpression or control was detected by TUNEL assay. Scale bars, 100 µm. I Cellular apoptosis of HepG2 cells with METTL16 overexpression or control was detected by TUNEL assay. Scale bars = 100 µm. J Cellular apoptosis of SNU-398 cells with METTL16 overexpression or control was detected by caspase-3 activity assay. K Cellular apoptosis of HepG2 cells with METTL16 overexpression or control was detected by caspase-3 activity assay. L Weight and photograph of subcutaneous tumors formed by SNU-398 cell with METTL16 overexpression or control. Results are shown as mean ± SD of n = 3 independent experiments ( B – K ) or n = 6 mice in each group ( L ). * P < 0.05, ** P < 0.01 by Student’s t -test ( B – K ) or Mann–Whitney test ( L )

Article Snippet: After incubation with primary antibodies against METTL16 (78 kDa, #17676, 1:1000, Cell Signaling Technology, Danvers, MA, USA) or GAPDH (36 kDa, 60004-1-Ig, 1:5000, Proteintech, Chicago, IL, USA) at 4 °C overnight, the membranes were further incubated with IRDye 680RD Goat anti-Mouse IgG Secondary Antibody (926-68070, 1:10000, Li-Cor Biosciences, Lincoln, NE, USA) or IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, 1:10000, Li-Cor).

Techniques: Over Expression, Expressing, Control, Western Blot, Migration, Transwell Migration Assay, Transwell Invasion Assay, EdU Assay, CCK-8 Assay, TUNEL Assay, Caspase-3 Activity Assay, MANN-WHITNEY

Journal: Cellular & Molecular Biology Letters

Article Title: METTL16 promotes hepatocellular carcinoma progression through downregulating RAB11B-AS1 in an m 6 A-dependent manner

doi: 10.1186/s11658-022-00342-8

Figure Lengend Snippet: The roles of METTL16 knockdown in HCC. A METTL16 protein expression in SNU-398 and HepG2 cells with METTL16 knockdown or control was detected by western blot. B Migration ability of SNU-398 and HepG2 cells with METTL16 knockdown or control was detected by transwell migration assay. Scale bars, 100 µm. C Invasion ability of SNU-398 and HepG2 cells with METTL16 knockdown or control was detected by transwell invasion assay. Scale bars, 100 µm. D Cellular proliferation of SNU-398 cells with METTL16 knockdown or control was detected by EdU assay. Scale bars, 100 µm. E Cellular proliferation of HepG2 cells with METTL16 knockdown or control was detected by EdU assay. Scale bars, 100 µm. F Cellular proliferation of SNU-398 cells with METTL16 knockdown or control was detected by CCK-8 assay. G Cellular proliferation of HepG2 cells with METTL16 knockdown or control was detected by CCK-8 assay. H Cellular apoptosis of SNU-398 cells with METTL16 knockdown or control was detected by TUNEL assay. Scale bars, 100 µm. I Cellular apoptosis of HepG2 cells with METTL16 knockdown or control was detected by TUNEL assay. Scale bars, 100 µm. J Cellular apoptosis of SNU-398 cells with METTL16 knockdown or control was detected by caspase-3 activity assay. K Cellular apoptosis of HepG2 cells with METTL16 knockdown or control was detected by caspase-3 activity assay. Results are shown as mean ± s.d. of n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA followed by Dunnett’s multiple comparisons test

Article Snippet: After incubation with primary antibodies against METTL16 (78 kDa, #17676, 1:1000, Cell Signaling Technology, Danvers, MA, USA) or GAPDH (36 kDa, 60004-1-Ig, 1:5000, Proteintech, Chicago, IL, USA) at 4 °C overnight, the membranes were further incubated with IRDye 680RD Goat anti-Mouse IgG Secondary Antibody (926-68070, 1:10000, Li-Cor Biosciences, Lincoln, NE, USA) or IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, 1:10000, Li-Cor).

Techniques: Knockdown, Expressing, Control, Western Blot, Migration, Transwell Migration Assay, Transwell Invasion Assay, EdU Assay, CCK-8 Assay, TUNEL Assay, Caspase-3 Activity Assay

Journal: Cellular & Molecular Biology Letters

Article Title: METTL16 promotes hepatocellular carcinoma progression through downregulating RAB11B-AS1 in an m 6 A-dependent manner

doi: 10.1186/s11658-022-00342-8

Figure Lengend Snippet: METTL16 downregulates RAB11B-AS1 through inducing m 6 A modification of RAB11B-AS1. A RAB11B-AS1 expression in SNU-398 and HepG2 cells with METTL16 overexpression or control was detected by RT-qPCR. B RAB11B-AS1 expression in SNU-398 and HepG2 cells with METTL16 knockdown or control was detected by RT-qPCR. C RIP assays were performed in SNU-398 and HepG2 cells to enrich the RNA bound by METTL16, followed by RT-qPCR to detect the enrichment of RAB11B-AS1. D CLIP assays were performed in SNU-398 and HepG2 cells to enrich the RNA directly bound by METTL16, followed by RT-qPCR to detect the enrichment of RAB11B-AS1. E MeRIP assays were performed in SNU-398 and HepG2 cells with METTL16 overexpression or control to enrich m 6 A modified RNA, followed by RT-qPCR to assess m 6 A modification level of RAB11B-AS1, MAT2A, and EEF1A1. F MeRIP assays were performed in SNU-398 and HepG2 cells with METTL16 knockdown or control to enrich m 6 A modified RNA, followed by RT-qPCR to assess m 6 A modification level of RAB11B-AS1, MAT2A, and EEF1A1. G The stability of RAB11B-AS1 over time was measured after blocking new RNA synthesis with α-amanitin (50 µM) in SNU-398 and HepG2 cells with METTL16 overexpression or control. H The stability of RAB11B-AS1 over time was measured after blocking new RNA synthesis with α-amanitin (50 µM) in SNU-398 and HepG2 cells with METTL16 knockdown or control. Results are shown as mean ± SD of n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student’s t -test ( A , C , D , E , G ) or one-way ANOVA followed by Dunnett’s multiple comparisons test ( B , F , H )

Article Snippet: After incubation with primary antibodies against METTL16 (78 kDa, #17676, 1:1000, Cell Signaling Technology, Danvers, MA, USA) or GAPDH (36 kDa, 60004-1-Ig, 1:5000, Proteintech, Chicago, IL, USA) at 4 °C overnight, the membranes were further incubated with IRDye 680RD Goat anti-Mouse IgG Secondary Antibody (926-68070, 1:10000, Li-Cor Biosciences, Lincoln, NE, USA) or IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, 1:10000, Li-Cor).

Techniques: Modification, Expressing, Over Expression, Control, Quantitative RT-PCR, Knockdown, Blocking Assay

Journal: Cellular & Molecular Biology Letters

Article Title: METTL16 promotes hepatocellular carcinoma progression through downregulating RAB11B-AS1 in an m 6 A-dependent manner

doi: 10.1186/s11658-022-00342-8

Figure Lengend Snippet: The expression pattern of RAB11B-AS1 in HCC and its association with METTL16. A RAB11B-AS1 expression in human HCC tissues ( n = 95) and normal liver tissues ( n = 39) from GSE45436 dataset. **** P < 0.0001 by Mann–Whitney test. B RAB11B-AS1 expression level in 63 pairs of HCC tissues and matched adjacent noncancerous liver tissues was measured by RT-qPCR. ** P < 0.01 by Wilcoxon signed-rank test. C Kaplan–Meier survival analysis of the correlation between RAB11B-AS1 expression and overall survival based on TCGA liver cancer data, analyzed by the online in silico tool Kaplan–Meier Plotter ( https://kmplot.com/analysis/ ). D Kaplan–Meier survival analysis of the correlation between RAB11B-AS1 expression and overall survival in our HCC cohort. n = 63 patients with HCC. P = 0.0171 by log-rank test. Median RAB11B-AS1 level was used as cutoff. E The correlation between RAB11B-AS1 and METTL16 expression in 374 HCC tissues according to TCGA dataset, analyzed by the online in silico tool ENCORI. F The correlation between RAB11B-AS1 and METTL16 expression in 63 HCC tissues detected by qRT-PCR. r = −0.2753, P = 0.029 by Spearman correlation analysis

Article Snippet: After incubation with primary antibodies against METTL16 (78 kDa, #17676, 1:1000, Cell Signaling Technology, Danvers, MA, USA) or GAPDH (36 kDa, 60004-1-Ig, 1:5000, Proteintech, Chicago, IL, USA) at 4 °C overnight, the membranes were further incubated with IRDye 680RD Goat anti-Mouse IgG Secondary Antibody (926-68070, 1:10000, Li-Cor Biosciences, Lincoln, NE, USA) or IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, 1:10000, Li-Cor).

Techniques: Expressing, MANN-WHITNEY, Quantitative RT-PCR, In Silico

Journal: Cellular & Molecular Biology Letters

Article Title: METTL16 promotes hepatocellular carcinoma progression through downregulating RAB11B-AS1 in an m 6 A-dependent manner

doi: 10.1186/s11658-022-00342-8

Figure Lengend Snippet: RAB11B-AS1 reverses the oncogenic roles of METTL16 in HCC. A Migration ability of SNU-398 cells with METTL16 and RAB11B-AS1 concurrent overexpression or control was detected by transwell migration assay. Scale bars, 100 µm. B Invasion ability of SNU-398 cells with METTL16 and RAB11B-AS1 concurrent overexpression or control was detected by transwell invasion assay. Scale bars, 100 µm. C Cellular proliferation of SNU-398 cells with METTL16 and RAB11B-AS1 concurrent overexpression or control was detected by EdU assay. Scale bars, 100 µm. D Cellular proliferation of SNU-398 cells with METTL16 and RAB11B-AS1 concurrent overexpression or control was detected by CCK-8 assay. E Cellular apoptosis of SNU-398 cells with METTL16 and RAB11B-AS1 concurrent overexpression or control was detected by TUNEL assay. Scale bars, 100 µm. F Cellular apoptosis of SNU-398 cells with METTL16 and RAB11B-AS1 concurrent overexpression or control was detected by caspase-3 activity assay. Results are shown as mean ± SD of n = 3 independent experiments. ** P < 0.01, *** P < 0.001; ns, not significant, by one-way ANOVA followed by Dunnett’s multiple comparisons test

Article Snippet: After incubation with primary antibodies against METTL16 (78 kDa, #17676, 1:1000, Cell Signaling Technology, Danvers, MA, USA) or GAPDH (36 kDa, 60004-1-Ig, 1:5000, Proteintech, Chicago, IL, USA) at 4 °C overnight, the membranes were further incubated with IRDye 680RD Goat anti-Mouse IgG Secondary Antibody (926-68070, 1:10000, Li-Cor Biosciences, Lincoln, NE, USA) or IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, 1:10000, Li-Cor).

Techniques: Migration, Over Expression, Control, Transwell Migration Assay, Transwell Invasion Assay, EdU Assay, CCK-8 Assay, TUNEL Assay, Caspase-3 Activity Assay

Journal: Biotechnology for Biofuels

Article Title: Improved cold tolerance in switchgrass by a novel CCCH-type zinc finger transcription factor gene, PvC3H72 , associated with ICE1–CBF–COR regulon and ABA-responsive genes

doi: 10.1186/s13068-019-1564-y

Figure Lengend Snippet: PvC3H72 subcellular localization and transcriptional activity assay. a Subcellular localization of PvC3H72–GFP fusion protein in Arabidopsis protoplast. GFP protein alone shows fluorescent signals in nucleus, membrane, and cytoplasm. DAPI is a staining dye for the nucleus. Bars = 5 µm. b PvC3H72 transcriptional activity in yeast system. GUS was used as a negative control. c Schematic diagram of three plasmids used for transactivation activity. Three plasmids (effector, reporter and internal control) were electroporated into Arabidopsis protoplasts at the ratio of 5:4:1. d PvC3H72 showed transactivation activity in the plant cells. All the experiments were repeated three times with similar results. Letters above bars indicate significant difference at P < 0.05 ( n = 3)

Article Snippet: DAPI were used to stain the nucleus, and the GFP signal were detected under a Zeiss LSM 780 laser scanning confocal microscope (Carl Zeiss SAS, Jena, Germany).

Techniques: Activity Assay, Membrane, Staining, Negative Control, Control