Journal: European Journal of Medical Research

Article Title: HDAC inhibition activates the apoptosome via Apaf1 upregulation in hepatocellular carcinoma

doi: 10.1186/s40001-016-0217-x

Figure Lengend Snippet: Expression of Apaf1 after siRNA transfection against HDAC1 – 3 in HLE and HLF. 48 h after transfection of siRNA against HDAC1 – 3 , the HCC cell lines a HLE and b HLF showed a significant increased expression of Apaf1 measured by qRTPCR. c Western blot analysis of Apaf1 after siRNA showed a significant increase in Apaf1 expression compared with controls. Values are mean ± SEM, n = 3. *** p < 0.001, ** p < 0.01, * p ≤ 0.05, ANOVA plus Dunnett ’s posttest

Article Snippet: The Agilent GeneSpring GX Data Analysis Software was used for bioinformatic analysis. mRNAs of interest were validated by qRTPCR as previously described [ ] with Taqman Assay [Hs00559421_m1, amplicon spans exon 10 and 11 with a length of 112 bp (Applied Biosystems)] and Western blot using antibody against Apaf1 [#8723 Cell Signaling/NEB Danvers, MA, USA, Antibody ID: AB_10829610 from http://www.antibodyregistry.org , used 1:1000, blocking with 3 % Slim Fast chocolate (Allpharm)].

Techniques: Expressing, Transfection, Western Blot

Journal: European Journal of Medical Research

Article Title: HDAC inhibition activates the apoptosome via Apaf1 upregulation in hepatocellular carcinoma

doi: 10.1186/s40001-016-0217-x

Figure Lengend Snippet: Expression of Apaf1 after 12-h treatment with the HDACi TSA. Expression of Apaf1 after 12-h treatment with the HDACi TSA in the HCC cell lines HLE, HLF, Huh7 and HepG2 measured by qRTPCR. After treatment, Apaf1 showed an increased expression compared with controls. Values are mean ± SEM, n = 3, * p ≤ 0.05, ANOVA plus Dunnett ’s posttest

Article Snippet: The Agilent GeneSpring GX Data Analysis Software was used for bioinformatic analysis. mRNAs of interest were validated by qRTPCR as previously described [ ] with Taqman Assay [Hs00559421_m1, amplicon spans exon 10 and 11 with a length of 112 bp (Applied Biosystems)] and Western blot using antibody against Apaf1 [#8723 Cell Signaling/NEB Danvers, MA, USA, Antibody ID: AB_10829610 from http://www.antibodyregistry.org , used 1:1000, blocking with 3 % Slim Fast chocolate (Allpharm)].

Techniques: Expressing

Journal: European Journal of Medical Research

Article Title: HDAC inhibition activates the apoptosome via Apaf1 upregulation in hepatocellular carcinoma

doi: 10.1186/s40001-016-0217-x

Figure Lengend Snippet: Expression of APAF1 measured by qRTPCR in 23 primary HCC compared to a human reference. Primary human HCC showed reduced expression of Apaf1 compared to a human reference

Article Snippet: The Agilent GeneSpring GX Data Analysis Software was used for bioinformatic analysis. mRNAs of interest were validated by qRTPCR as previously described [ ] with Taqman Assay [Hs00559421_m1, amplicon spans exon 10 and 11 with a length of 112 bp (Applied Biosystems)] and Western blot using antibody against Apaf1 [#8723 Cell Signaling/NEB Danvers, MA, USA, Antibody ID: AB_10829610 from http://www.antibodyregistry.org , used 1:1000, blocking with 3 % Slim Fast chocolate (Allpharm)].

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: Impact of the modulation of TYRO3 expression on the radiosensitivity of bladder cancer cell lines. ( a – c ) Representative clonogenic survival curves of RT112 and 5637 cell lines ( upper panel). 48 h after transfection with siLUC (control, red), siTYRO3#4 (blue), siTYRO3#801 (green) or diluted concentration of siRNA#1 (black) cells were exposed to increased doses of gamma-rays. In each case, downregulation was confirmed by western blot at 48 h after transfection. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( d ) Representative survival curves of UM-UC-3 control (empty plasmid, red) and TYRO3 over-expressed cell lines (TYRO3 encoding- plasmid, purple) ( upper panel). The upregulation was confirmed by western blot. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( e ) Phosphorylated TYRO3 shown in western blot ( lower panel) after immunoprecipitation of the cell extracts with phospho-Tyrosine antibodies and its relative expression in 5637 cells quantified using Image-J software and normalized to that of total TYRO3 ( upper panel). Data represents mean ± SD of 3 independent experiments. Unpaired t-test analysis: * p < 0.05; ** p < 0.005, ns: not significant.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Expressing, Transfection, Control, Concentration Assay, Western Blot, Plasmid Preparation, Immunoprecipitation, Software

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on Ionizing Radiation-Induced Foci and DNA damage. γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 downregulated RT112 ( a ) or 5637 ( c ) cells (scale bar 5 microns); Quantification of cells containing more than 10 γH2AX foci at 24 h after 2 Gy of irradiation in the downregulated RT112 ( b ) and 5637 ( d ) cell lines; ( e ) γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 overexpressing UM-UC-3 cell line (Scale bar 5 microns); ( f ) Quantification of cells containing more than 10 γH2AX foci at 30 min and 24 h after 2 Gy of irradiation in TYRO3 overexpressing cell lines. The data shown above is from three different experiments and error bars represent the SD. Unpaired t -test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0005, ns: non-significant. Representative images of the alkaline Comet assay performed on TYRO3 downregulated RT112 ( g ) and 5637 ( h ) cells and the resulting Olive tail moments analysis in TYRO3 downregulated RT112 ( i ) and 5637 ( j ) cells irradiated at 6 Gy. The data shown here is from three independent experiments analyzing 200 nucleus per condition per experiment, horizontal bars represent the median values. Kruskal-Wallis nonparametric tests with Multiple comparisons were used: * p < 0.05; ** p < 0.005, ns: non-significant; ( k ) western blot of DNA damage repair proteins; TYRO3 was downregulated, irradiated at 6 Gy and the lysates were prepared and analyzed 0.5–24 h after.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Alkaline Single Cell Gel Electrophoresis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on DNA damage response pathways. ( a ) Volcano plot showing the fold change (Log2 Ratio) versus negative log of the p-value of differentially expressed genes after Nanostring analysis between BCa (RT112 and 5637) 6 Gy irradiated cells versus BCa 6 Gy irradiated cells after complete TYRO3 knock-down (siTYRO3#4 and siTYRO3#801). Significant: p -value < 0.05 ( b ) Log2 ratio of the significantly expressed genes ( p < 0.05) after Nanostring analysis between the two groups. The name of the up- or downregulated genes are listed on the left. On the right are the corresponding Nanostring gene annotations. ( c ) Protein-protein association network of the up and downregulated genes assessed using the STRING database.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 downregulation affects cell cycle following irradiation. Analysis of the cell cycle distribution 24 h after 6 Gy irradiation on TYRO3-downregulated RT112 ( a ) and 5637 ( b ) cells. Comparison of percentage (%) of cells in G2/M in RT112 ( c ) and 5637 ( d ) cells. Data shown is from three different experiments and error bars represent the SD. Unpaired t-test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005, ns: non-significant. ( e ) western blot of cell cycle proteins 30 min and up to 24 h after irradiation.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Comparison, Western Blot

Journal: eLife

Article Title: Siglec1-expressing subcapsular sinus macrophages provide soil for melanoma lymph node metastasis

doi: 10.7554/eLife.48916

Figure Lengend Snippet:

Article Snippet: Antibody , Cy3 AffiniPure Goat Anti-Rat IgG (H+L) , Jackson ImmunoResearch , Cat# 112-165-167, RRID: AB_2338251 , (1:500).

Techniques: Control, Adsorption, Plasmid Preparation, Software, Recombinant, Expressing, Transfection, Construct, Negative Control, Sequencing

Journal: iScience

Article Title: Mice carrying the full-length human immunoglobulin loci produce antigen-specific human antibodies with the lambda light chain

doi: 10.1016/j.isci.2024.111258

Figure Lengend Snippet:

Article Snippet: Anti-Igλ, Human, Goat-Poly, HRP , Bethyl Laboratories , Cat# A80-116P; RRID: AB_67591.

Techniques: Activation Assay, Marker, Virus, Recombinant, Transfection, Adjuvant, Enzyme-linked Immunosorbent Assay, Expressing, Knock-Out, cDNA Synthesis, Plasmid Preparation, Software, Fluorescence, Imaging

Journal: eLife

Article Title: Sushi domain-containing protein 4 controls synaptic plasticity and motor learning

doi: 10.7554/eLife.65712

Figure Lengend Snippet:

Article Snippet: Antibody , Goat polyclonal anti-rat HRP , Jackson Immune Research Laboratories , #112-035-175 , (1:10,000).

Techniques: Knock-Out, Expressing, Recombinant, Software, Plasmid Preparation

Journal: Regenerative Therapy

Article Title: Preparation of iPS cell-derived CD31 + endothelial cells using three-dimensional suspension culture

doi: 10.1016/j.reth.2018.06.004

Figure Lengend Snippet: Characterisation of hiPS cell-derived CD31 + cells. (A) Endothelial marker gene expression in differentiated hiPS cells. hiPS cell differentiation was performed in 30- or 100-ml culture vessels. (B) Mean percentage of cells expressing CD31 on day 9 of differentiation from KDR + or KDR − cells. After 7 days of differentiation, both KDR + and KDR − cells were isolated by MACS, re-cultured onto collagen IV-coated tissue culture dishes, and immunostained with CD31 antibodies. Scale bars = 400 pm. CD31 + cell number was analysed using MetaXpress software. (C) Phase-contrast image of hiPS cell-derived CD31 + cells. Magnification, ×40. (D) Network formation by induced CD31 + cells after 24 h of culture on top of Matrigel. Magnification, ×40. (E) Comparison of endothelial marker gene expression between hiPS cell-derived CD31 + cells and tissue-derived vascular endothelial cells. The expression levels of genes were analysed by Taqman gene expression assay and gene expression was normalised to endogenous β-actin. (F) Immunostaining of CD31 + cells derived from hiPS cells on day 13 of differentiation. CD31 (left panel, red) and vascular endothelial cadherin (right panel, green). (B-F) hiPS cell differentiation from day 0 to day 7 was performed in 30-ml culture vessels. Nuclei were stained with Hoechst 33342 (blue) in (B) and (F). Scale bars = 400 pm in (B) and (F). Values are shown as mean ± standard deviation for at least three separate experiments in (A), (B), (E) and (F). Asterisk indicates statistically significant difference ( p < 0.05). NS, not significant.

Article Snippet: Phycoerythrin-conjugated monoclonal antibodies for human vascular endothelial (VE)-cadherin (R&D Systems) and monoclonal antibodies for human CD31 conjugated with phycoerythrin were used for immunocytochemistry.

Techniques: Derivative Assay, Marker, Gene Expression, Cell Differentiation, Expressing, Isolation, Cell Culture, Software, Comparison, Immunostaining, Staining, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Unraveling AURKB as a potential therapeutic target in pulmonary hypertension using integrated transcriptomic analysis and pre-clinical studies

doi: 10.1016/j.xcrm.2025.101964

Figure Lengend Snippet: Therapeutic effects of barasertib in male rats exposed to Sugen/hypoxia (A) Study design using the Sugen/hypoxia (Su/Hx) rat model. (B) Pulmonary artery acceleration time (PAAT), right ventricular fractional area change (RVFAC), tricuspid annular plane systolic excursion (TAPSE), S wave, stroke volume (SV), and cardiac output (CO) determined by echocardiography at the end of the protocol in control, Su/Hx+Veh, and Su/Hx+barasertib male rats ( n = 4–9/group). (C) Effect of AURKB inhibition on right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP), as assessed by right heart catheterization at the end of the protocol ( n = 4–9/group). (D) Representative images of distal PAs stained with Elastica van Gieson (EVG) and quantification of vascular remodeling in control, Su/Hx+Veh, and Su/Hx+barasertib rats ( n = 4–9/group). (E) Representative images of distal PAs labeled with proliferating cell nuclear antigen (PCNA, proliferative marker, red), p16, or p21 ( n = 4–9/group). PASMCs were labeled with alpha smooth muscle actin (αSMA, green). The quantifications of the percentage of PASMCs positive for PCNA, p16, or p21 are shown. Scale bars, 20 μm. Scatter dot plots show individual values and mean ± SEM. Assessment of the normality of the data was performed using Shapiro-Wilk test. Statistical analyses were performed using one-way ANOVA or Kruskal-Wallis’s test followed by Dunnett’s post hoc test; ∗ p < 0.05; ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figures S8 and .

Article Snippet: Smooth muscle cell basal medium , Cell Applications , Cat# 310-500.

Techniques: Control, Inhibition, Staining, Labeling, Marker

Journal: Cell Reports Medicine

Article Title: Unraveling AURKB as a potential therapeutic target in pulmonary hypertension using integrated transcriptomic analysis and pre-clinical studies

doi: 10.1016/j.xcrm.2025.101964

Figure Lengend Snippet: Barasertib reduces vascular remodeling in human precision-cut lung slices (A) Experimental setup for precision-cut lung slices (PCLSs) from control, patients with PAH, and patients with idiopathic pulmonary fibrosis. (B) Representative images of distal PAs stained with Elastica van Gieson (EVG) or labeled with proliferating cell nuclear antigen (PCNA) or p21 in PCLSs prepared from control patients ( n = 5) after exposure or not to a growth factor cocktail (GF, FGF2 + PDGF-BB + ET1) in presence or not to barasertib for 10 days. PASMCs were labeled with alpha smooth muscle actin (αSMA, green). The quantification of vascular remodeling and PASMCs positive for PCNA or p21 is shown. (C) Representative images of distal PAs stained with EVG or labeled with PCNA or p21 in PCLSs from patients with PAH ( n = 6). (D) Representative images of distal PAs stained with EVG or labeled with PCNA or p21 in PCLSs from patients with IPF complicated with pulmonary hypertension (PH) ( n = 2). For each experiment, the quantifications of vascular remodeling and PCNA- or p21-positive PASMCs (average of 40–45 arteries per patient) are shown. Scale bars, 25 μm. Values are represented as means ± SEM. Assessment of the normality of the data was performed using Shapiro-Wilk test. Statistical analyses were performed using repeated measures one-way ANOVA test followed by Dunnett’s post hoc test or paired Student’s t test; ∗ p < 0.05; ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Smooth muscle cell basal medium , Cell Applications , Cat# 310-500.

Techniques: Control, Staining, Labeling

Journal: Cell Reports Medicine

Article Title: Unraveling AURKB as a potential therapeutic target in pulmonary hypertension using integrated transcriptomic analysis and pre-clinical studies

doi: 10.1016/j.xcrm.2025.101964

Figure Lengend Snippet:

Article Snippet: Smooth muscle cell basal medium , Cell Applications , Cat# 310-500.

Techniques: Virus, Plasmid Preparation, Recombinant, Negative Control, Protease Inhibitor, Staining, EdU Assay, TUNEL Assay, Western Blot, SYBR Green Assay, Chromatin Immunoprecipitation, Magnetic Beads, RNA Sequencing Assay, Expressing, Software

Journal: EMBO Reports

Article Title: Auditory fear memory retrieval requires BLA-LS and LS-VMH circuitries via GABAergic and dopaminergic neurons

doi: 10.1038/s44319-025-00403-x

Figure Lengend Snippet: ( A ) A schematic of the experimental design. Mice were sacrificed for the detection of c-Fos positive cells in the LS at 1 h after memory retrieval tone stimuli. ( B ) Representatives of immunofluorescent staining showing co-localization of GAD65&67 or TH with c-Fos in the LS, respectively. The white arrows indicate GABAergic and dopaminergic cells in the LS. GAD65&67, glutamate decarboxylase 65 & glutamate decarboxylase 67; TH, tyrosine hydroxylase; MAP2, microtubule-associated protein 2. ( C ) Schematic of DLAG, L-AMPT or vehicle injection and experimental timeline for examining the role of GABAergic and dopaminergic cells in the LS in fear conditioning. Mice were sacrificed for the detection of c-Fos-positive cells in the LS at 1 h after memory retrieval tone stimuli. DLAG DL-2-Allylglycine, L-AMPT α -Methyl-L-tyrosine. ( D ) Representatives of immunofluorescent staining showing co-localization of NeuN with c-Fos in the LS. The dashed lines indicate the boundary of the LS. SHi septohippocampal nucleus, cc corpus callosum, LV lateral ventricle, NeuN Neuronal nuclear protein. ( E ) Left panel: quantification of c-Fos expression [ n = 10 mice per group; H = 30.029 (df = 3) on rank, P < 0.0001; Kruskal–Wallis test]. Right panel: performance during fear conditioning [ n = 10 mice per group; F (3,36) = 11.40, P < 0.0001; Brown–Forsythe and Welch one-way analysis of variance test]. Data are presented as mean ± SEM (normal distribution data) or median ± interquartile range (not normal distribution data) with the presentation of individual animal data in the bar graph. .

Article Snippet: The primary antibodies used were: anti-c-Fos (1:1000, rabbit, Abcam, catalog number: ab190289); anti-vGluT1 antibody (1:5000, mouse, Abcam, catalog number: ab 242204), anti-GAD65&67 antibody (1:200, rabbit, Abcam, catalog number: ab 11070); anti-tyrosine hydroxylase antibody (1:750, rabbit, Abcam, catalog number: ab 112, or 1:200, mouse, Cell Signaling, catalog number: 45648); anti-GABA (1:500, rabbit, MilliporeSigma, catalog number: A2052), anti-microtubule-associated protein 2 (MAP2) antibody (1:200, mouse, Abcam, catalog number: ab 11267 and 1:200, rabbit, Abcam, catalog number: ab 32454); anti-neuronal nuclear protein (NeuN) antibody (1:1000, rabbit, Abcam, catalog number: ab128886); anti-orexin A antibody (1:1000, rabbit, Abcam, catalog number: ab6214); anti-orexin B antibody (1:100, mouse, Abcam, catalog number: ab89888); and anti-Cre recombinase antibody (1:1000, rabbit, Abcam, catalog number: ab216262, or 1:200, rabbit, Cell Signaling, catalog number: 15036s).

Techniques: Staining, Injection, Expressing

Journal: EMBO Reports

Article Title: Auditory fear memory retrieval requires BLA-LS and LS-VMH circuitries via GABAergic and dopaminergic neurons

doi: 10.1038/s44319-025-00403-x

Figure Lengend Snippet: ( A ) A schematic of the viral injection. Mice were used in tone-related fear conditioning test and sacrificed for the detection of c-Fos positive cells in the LS at 1 h after memory retrieval tone stimuli. ( B ) Representatives of immunofluorescent staining showing co-localization of Cre with mCherry (HM4Di) in the LS. The white arrows indicate co-staining cells in the LS. LV lateral ventricle. ( C ) Performance during tone-related memory phase [ n = 6–10 mice per group; F (4,35) = 3.58, P = 0.015; one-way analysis of variance test followed with t test]. Data are presented as mean ± EM (normal distribution data) with the presentation of individual animal data in the bar graph. C21 compound 21, GAD glutamate decarboxylase, TH tyrosine hydroxylase, V vehicle. ( D ) Quantification of c-Fos expression [ n = 6 mice per group; F (4,25) = 15.39, P < 0.0001; one-way analysis of variance test]. Data are presented as mean ± SEM (normal distribution data) with the presentation of individual animal data in the bar graph. ( E ) Representatives of immunofluorescent staining showing c-Fos staining in the LS. LV lateral ventricle. .

Article Snippet: The primary antibodies used were: anti-c-Fos (1:1000, rabbit, Abcam, catalog number: ab190289); anti-vGluT1 antibody (1:5000, mouse, Abcam, catalog number: ab 242204), anti-GAD65&67 antibody (1:200, rabbit, Abcam, catalog number: ab 11070); anti-tyrosine hydroxylase antibody (1:750, rabbit, Abcam, catalog number: ab 112, or 1:200, mouse, Cell Signaling, catalog number: 45648); anti-GABA (1:500, rabbit, MilliporeSigma, catalog number: A2052), anti-microtubule-associated protein 2 (MAP2) antibody (1:200, mouse, Abcam, catalog number: ab 11267 and 1:200, rabbit, Abcam, catalog number: ab 32454); anti-neuronal nuclear protein (NeuN) antibody (1:1000, rabbit, Abcam, catalog number: ab128886); anti-orexin A antibody (1:1000, rabbit, Abcam, catalog number: ab6214); anti-orexin B antibody (1:100, mouse, Abcam, catalog number: ab89888); and anti-Cre recombinase antibody (1:1000, rabbit, Abcam, catalog number: ab216262, or 1:200, rabbit, Cell Signaling, catalog number: 15036s).

Techniques: Injection, Staining, Expressing

Journal: EMBO Reports

Article Title: Auditory fear memory retrieval requires BLA-LS and LS-VMH circuitries via GABAergic and dopaminergic neurons

doi: 10.1038/s44319-025-00403-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The primary antibodies used were: anti-c-Fos (1:1000, rabbit, Abcam, catalog number: ab190289); anti-vGluT1 antibody (1:5000, mouse, Abcam, catalog number: ab 242204), anti-GAD65&67 antibody (1:200, rabbit, Abcam, catalog number: ab 11070); anti-tyrosine hydroxylase antibody (1:750, rabbit, Abcam, catalog number: ab 112, or 1:200, mouse, Cell Signaling, catalog number: 45648); anti-GABA (1:500, rabbit, MilliporeSigma, catalog number: A2052), anti-microtubule-associated protein 2 (MAP2) antibody (1:200, mouse, Abcam, catalog number: ab 11267 and 1:200, rabbit, Abcam, catalog number: ab 32454); anti-neuronal nuclear protein (NeuN) antibody (1:1000, rabbit, Abcam, catalog number: ab128886); anti-orexin A antibody (1:1000, rabbit, Abcam, catalog number: ab6214); anti-orexin B antibody (1:100, mouse, Abcam, catalog number: ab89888); and anti-Cre recombinase antibody (1:1000, rabbit, Abcam, catalog number: ab216262, or 1:200, rabbit, Cell Signaling, catalog number: 15036s).

Techniques: Virus, Software

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 expression is upregulated in LUAD, and its increased expression is associated with unfavorable OS and FP. (A) METTL1 expression profile in LUAD tissues (n=483) and normal lung tissues (n=59) from TCGA database was analyzed using Gene Expression Profiling Interactive Analysis software. METTL1 expression was significantly upregulated in LUAD tissues. (B) METTL1 expression was significantly higher in different LUAD stages compared with normal tissues. No significant differences were observed among the different tumor stages. The METTL1 expression profile in different LUAD stages (stage I, n=277; stage II, n=125; stage III, n=85 and stage IV, n=28) and normal lung tissues (n=59) from TCGA database was analyzed using UALCAN software. (C) The METTL1 expression profile in LUAD tissues (n=58) and normal lung tissues (n=49) from the GSE10072 dataset in the GEO database was analyzed using GraphPad Prism 5.0 software. METTL1 expression was significantly upregulated in LUAD tissues. (D) METTL1 expression was significantly higher in different LUAD stages compared with normal tissues. No significant differences were observed among the different stages. The METTL1 expression profile in different stages (stage I, n=22; stage II, n=21; stage III, n=12 and stage IV, n=3) and normal lung tissues (n=49) from the GSE10072 dataset in the GEO database was analyzed using GraphPad Prism 5.0 software. (E) METTL1 expression was upregulated in LUAD tissues. Representative images of immunohistochemistry staining from the Human Protein Atlas database. Scale bar at low magnification, 200 µM; high magnification, 50 µM. HPA Patients’ ID, negative: 2268, medium: 3052, low: 2403. (F) High METTL1 expression was associated with unfavorable OS. The OS curve of 719 patients was plotted [cut-off value, 347; HR=1.7 (1.35–2.15), 95% CI; log-rank P=6×10 −6 −05]. (G) High METTL1 expression was associated with unfavorable FP. The FP curve of 461 patients was plotted [cut-off value, 324; HR=2.26 (1.65–3.1), 95% CI; log-rank P=2.2×10 −7 ]. The online Kaplan Meier Plotter software was used to construct the OS and FP graphs. *P<0.05, ***P<0.001. METTL1, methyltransferase-like 1; LUAD, lung adenocarcinoma; OS, overall survival; FP, first progression; TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus; HR, hazard ratio; CI, confidence interval.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing, Gene Expression, Software, Immunohistochemistry, Staining, Construct

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: Association between METTL1 expression and the clinicopathological characteristics of patients with lung adenocarcinoma (n=517).

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: Cox regression multivariate analysis of METTL1 on OS and FP of patients with lung adenocarcinoma.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques:

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 promotes colony formation and A549 cell proliferation. (A) METTL1 expression was increased in A549 cells. (B) Western blot analysis demonstrated that METTL1 protein was upregulated and downregulated in the overexpression and knockdown experiments, respectively. Overexpression of METTL1 promoted A549 and H1993 cell (C) proliferation and (D) colony formation. METTL1 silencing attenuated A549 and H1993 cell (C) proliferation and (D) colony formation. *P<0.05, **P<0.01, ***P<0.001. METTL1, methyltransferase-like 1; si, small interfering; OD, optical density.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing, Western Blot, Over Expression, Knockdown

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 inhibits autophagy in A549 cells. (A) METTL1 overexpression inhibited the conversion of LC3-I to LC3-II and the degradation of p62/SQSTM1, suggesting that METTL1 overexpression may inhibit autophagy. (B) METTL1 knockdown enhanced the conversion of LC3-I to LC3-II and the degradation of p62/SQSTM1, suggesting that METTL1 knockdown may promote autophagy. (C) METTL1 knockdown increased both the size and number of GFP-LC3 puncta in HCC827/GFP-LC3 cells, suggesting that METTL1 knockdown may promote autophagy. *P<0.05, **P<0.01, ***P<0.001. METTL1, methyltransferase-like 1; SQSTM1, sequestosome 1; si, small interfering.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Over Expression, Knockdown

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 activates the AKT/mTORC1 signaling pathway. GSEA analysis of the TCGA-LUAD dataset revealed that the gene sets (A) HALLMARK_MTORC1_SIGNALING and (B) HALLMARK_PI3K_AKT_MTOR_SIGNALING were enriched in the high-METTL1 expression group. (C) DAVID analysis of the GSE112180 dataset indicated that the gene cluster of the PI3K/AKT signaling pathway was enriched in the high-METTL1 expression group (D) METTL1 overexpression increased the protein levels of p-AKT and p-S6K, the effects of which were reversed following METTL1 silencing. Total protein levels of AKT and S6K remained unchanged compared with the control group. *P<0.05. METTL1, methyltransferase-like 1; p-AKT, phosphorylated protein kinase B; mTORC1, mechanistic target of rapamycin complex 1; GSEA, Gene Set Enrichment Analysis; TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; DAVID, Database for Annotation, Visualization and Integrated Discovery; PI3K, phosphatidylinositol 3-kinase; si, small interfering.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing, Over Expression, Control