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: Journal of Cellular and Molecular Medicine

Article Title: Effects of the multi‐kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia

doi: 10.1111/jcmm.14927

Figure Lengend Snippet: Comparison of effects of FLT3 inhibitors against the growth of human AML cell lines. Cells were treated for approximately three days. Targeted SYK inhibitor, PRT062607, was tested in parallel with the FLT3 inhibitors. (A‐F) Treatment of AML cell lines with midostaurin (A), gilteritinib (B), sorafenib (C), crenolanib (D), quizartinib (E) and PRT062607 (F)

Article Snippet: 80 mg/kg was the tolerated dose for midostaurin purchased from MedChemExpress, LC Labs and BOC Sciences, whereas 100 mg/kg was the tolerated dose for midostaurin received from Novartis Pharma AG.

Techniques: Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Effects of the multi‐kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia

doi: 10.1111/jcmm.14927

Figure Lengend Snippet: Effects of midostaurin on signalling molecules in wt FLT3‐expressing and mutant FLT3‐expressing cells. (A) Effects of midostaurin at 100 nmol/L on levels of Bcl‐xL, Bcl‐2 and Mcl‐1 in mutant FLT3‐expressing MOLM14 cells. (B, C) Effects of midostaurin on levels of Bcl‐xL, Bcl‐2 and Mcl‐1 in wt FLT3‐expressing Kasumi‐1‐luc+ cells (B) or OCI‐AML2 cells (C). (D) Induction of apoptosis by midostaurin in wt FLT3‐expressing AML cells. The apoptosis studies carried out for midostaurin‐treated Kasumi‐1‐luc+ cells and OCI‐AML2 cells were performed once each. The flow cytometry data from which the numerical values shown in the bar graphs were derived are now shown in Figure . (E) Effects of midostaurin at the indicated concentrations on phosphorylation of key molecules involved in PI3K/AKT and MAPK signalling in wt FLT3‐expressing Kasumi‐1‐luc+ cells. (F) Effects of midostaurin at the indicated concentrations on phosphorylation of S6 in wt FLT3‐expressing AML cell lines conferring differential sensitivity to midostaurin. WB = Western blot

Article Snippet: 80 mg/kg was the tolerated dose for midostaurin purchased from MedChemExpress, LC Labs and BOC Sciences, whereas 100 mg/kg was the tolerated dose for midostaurin received from Novartis Pharma AG.

Techniques: Expressing, Mutagenesis, Flow Cytometry, Derivative Assay, Phospho-proteomics, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Effects of the multi‐kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia

doi: 10.1111/jcmm.14927

Figure Lengend Snippet: In vivo effects of midostaurin on growth of leukaemia cells in an SKNO‐1‐luc+ xenograft model and in an OCI‐AML3‐luc+ xenograft model. (A) Measure of leukaemia burden in vehicle‐ versus midostaurin‐treated mice for SKNO‐1‐luc+ xenograft model. For total flux bioluminescence, the Mann‐Whitney test (two‐tailed) was carried out. Vehicle versus midostaurin, P < .0001 (Day 8); P < .0001 (Day 15); P < .0001 (Day 22); P < .0001 (Day 29). (B) Measure of survival of vehicle‐ versus midostaurin‐treated mice for SKNO‐1‐luc+ xenograft model. The log‐rank (Mantel‐Cox) test and the Gehan‐Breslow‐Wilcoxon tests were carried out for survival curve comparisons. For both tests, P < .0001. (C) Effects of midostaurin in vivo against mice harbouring SKNO‐1‐luc+ cells: supine and prone (high scale). Days 22‐36. Representative images (n = 5). (D) Measure of leukaemia burden in vehicle‐ versus midostaurin‐treated mice for OCI‐AML3‐luc+ xenograft model. For total flux bioluminescence, the Mann‐Whitney test (two‐tailed) was carried out. Vehicle versus midostaurin (80 mg/kg, Day 15), P = 0656; vehicle versus midostaurin (100 mg/kg, Day 15), P < .0001; midostaurin (80 mg/kg, Day 15) versus midostaurin (100 mg/kg, Day 15), P = .0001. (E) Measure of survival of vehicle‐ versus midostaurin‐treated mice for OCI‐AML3‐luc+ xenograft model. The log‐rank (Mantel‐Cox) test and the Gehan‐Breslow‐Wilcoxon tests were carried out for survival curve comparisons. For both tests, P < .0001. For vehicle versus midostaurin (80 mg/kg), the log‐rank (Mantel‐Cox) test yielded a P value = .0547, and the Gehan‐Breslow‐Wilcoxon test yielded a P value = .0210. For midostaurin (80 mg/kg) versus midostaurin (100 mg/kg), the log‐rank (Mantel‐Cox) test yielded a P value = .0027, and the Gehan‐Breslow‐Wilcoxon test yielded a P value = .0025. For vehicle versus midostaurin (100 mg/kg), the log‐rank (Mantel‐Cox) test yielded a P value = .0001, and the Gehan‐Breslow‐Wilcoxon test yielded a P value = .0002. (F) Effects of midostaurin in vivo against mice harbouring OCI‐AML3‐luc+ cells. Supine and prone (high scale), Days 8, 15 and 22. Representative images (n = 4)

Article Snippet: 80 mg/kg was the tolerated dose for midostaurin purchased from MedChemExpress, LC Labs and BOC Sciences, whereas 100 mg/kg was the tolerated dose for midostaurin received from Novartis Pharma AG.

Techniques: In Vivo, MANN-WHITNEY, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Effects of the multi‐kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia

doi: 10.1111/jcmm.14927

Figure Lengend Snippet: Comparison of effects of FLT3 inhibitors on growth of cells expressing activated SYK or oncogenic FLT3. (A‐C) Proliferation curves generated for parental Ba/F3 cells, Ba/F3‐SYK‐TEL cells and Ba/F3‐FLT3‐ITD cells treated for approximately three days with FLT3 inhibitors (crenolanib, quizartinib, midostaurin, sorafenib and gilteritinib) or the targeted SYK inhibitor, PRT062607. (D) Comparison of effects of FLT3 inhibitors on activation of signalling molecules downstream of activated SYK. PRT062607 was included as a positive control for SYK inhibition

Article Snippet: 80 mg/kg was the tolerated dose for midostaurin purchased from MedChemExpress, LC Labs and BOC Sciences, whereas 100 mg/kg was the tolerated dose for midostaurin received from Novartis Pharma AG.

Techniques: Comparison, Expressing, Generated, Activation Assay, Positive Control, Inhibition

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: 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: Virus research

Article Title: Targeting HDAC11 activity by FT895 restricts EV71 replication.

doi: 10.1016/j.virusres.2023.199108

Figure Lengend Snippet: Fig. 4. FT895 suppresses EV71 replication via reducing HDAC11 activity and increasing AcH3 levels The anti-EV71 effect of FT895 in HeLa cells was employed by measuring viral protein expression, viral RNA levels and viral titers, respectively. (A) EV71 VP1 protein expression was measured by Western blotting. (B) EV71 VP1 RNA levels were analyzed by qPCR analysis. (C) The cell supernatants were harvested, and virus titers were assessed by virus titers assay. (D) The effect of FT895 (1 µM) treatment of HeLa cells in the absence or in the presence of EV71 BrCr for 24 h. After then, HDAC11 protein and AcH3 protein were determined by Western blotting assay. Western blot analysis of AcH3 and HDAC11. The bar graph shows densitometric quantification of HDAC11 and AcH3 levels. Data were normalized to the control. GAPDH was considered as the loading control. Data were obtained from three independent experiments. (E) The extracellular viral titers from A were measured by plague-forming assay. (F) HeLa cells were transfected with the plasmids shNC and shHDAC11 for 24 h; After transfection, they were both treated with FT895 (1 µM) for 2 h and then they were infected with EV71 at an MOI of 1 for 24 h. Western blotting analysis of VP1 and HDAC11 protein expression from the groups shNC and shHDAC11, respectively. **P < 0.01 and ***P < 0.001.

Article Snippet: FT895 (HY-112,285) was obtained from MCE (Med Chem Express) and dissolved in DMSO.

Techniques: Activity Assay, Expressing, Western Blot, Virus, Control, Transfection, Infection

Journal: Virus research

Article Title: Targeting HDAC11 activity by FT895 restricts EV71 replication.

doi: 10.1016/j.virusres.2023.199108

Figure Lengend Snippet: Fig. 5. FT895 administration impaired EV71 replication in vivo FT895 impairs EV71 replication in small intestine tissues in mice. 2-week-old C57BL/6 mice were intravenously injected with 1 × 106 PFU of Gluc-EV71 per mouse and then they randomly divided into 2 groups (n = 5 mice for each group) and 2 h later intraperitoneally administered with an HDAC11 inhibitor FT895 (50 mg/kg body weight) or equal volume of PBS as a negative control per day for 4 consecutive days. The mice were finally euthanized at 7 dpi and used for in vivo imaging studies. (A) In vivo imaging was performed on an IVIS 200 imaging system and images were further analyzed using Living Image software. The fluorescence intensity (pH/sec/cm2) summary of each mouse in (A) was quantified and presented as the mean ± SEM. (B) Viral loads in the small intestine tissues were observed in vivo imaging and the fluorescence intensity (pH/sec/cm2) summary of each intestine in (B) was quantified and presented as the mean ± SEM. (C) Viral loads in the small intestine tissues were quantified by qPCR analysis. (D) Viral loads in the feces were measured by plague-forming assay. (E) IL-10 mRNA expression in the small intestine tissues were quantified by qPCR analysis. The error bars indicate the means ± standard errors of the means (SEM). Data are representative of two inde pendent experiments. (**, P <0.01). All experiments were repeated at least three times with consistent results.

Article Snippet: FT895 (HY-112,285) was obtained from MCE (Med Chem Express) and dissolved in DMSO.

Techniques: In Vivo, Injection, Negative Control, In Vivo Imaging, Imaging, Software, Fluorescence, Expressing

Journal: British Journal of Cancer

Article Title: Targeting PIM kinase enhances the activity of sunitinib in renal cell carcinoma

doi: 10.1038/bjc.2011.426

Figure Lengend Snippet: PIM-1 is overexpressed in RCC cell lines and inhibition of PIM kinase activity with SGI-1776 promotes apoptosis and reduced viability. ( A ) PIM-1 kinase expression in RCC cell lines. Immunoblotting demonstrates elevated PIM-1 levels compared with normal RPTECs. Band intensity was quantified using ImageJ software. ( B ) SGI-1776 reduces RCC cell viability. Five RCC cell lines and RPTEC cells were incubated with the indicated concentrations of SGI-1776 for 72 h and cell viability was measured by MTT assay. Mean±s.d., n =3. ( C ) SGI-1776 reduces RCC clonogenic survival. Cells were treated with indicated concentrations of SGI-1776 for 24 h, washed and incubated in fresh media for 10 days. Colonies were fixed, stained with crystal violet and quantified. Mean±s.d., n =3. * Indicates a significant difference compared with control. P <0.05. ( D ) SGI-1776 decreases Bad phosphorylation at Ser112. Cells were treated with SGI-1776 for 48 h and phospho- and total Bad levels were determined by immunoblotting. Phospho-Bad band intensity was quantified using ImageJ software. ( E ) SGI-1776 induces apoptosis in RCC cell lines. Cells were treated with SGI-1776 for 48 h and apoptosis was measured by PI-FACS analysis. Mean±s.d., n =3. * Indicates a significant difference compared with control. P <0.05.

Article Snippet: Antibodies were obtained from the following commercial sources: anti-PIM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-Bad (Ser112) and Bad (Cell Signaling, Danvers, MA, USA); anti-actin and tubulin (Sigma-Aldrich, St Louis, MO, USA); anti-c-Myc, phospho-c-Myc (Ser62), and Bad (immunohistochemistry) (Novus Biologicals, Littleton, CO, USA); antiproliferating cell nuclear antigen (PCNA) (Dako, Glostrup, Denmark); goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratories, West Grove, PA, USA); Rat anti-mouse IgG2a-HRP (Serotec, Raleigh, NC, USA); and sheep anti-mouse-HRP and donkey anti-rabbit-HRP (Amersham, Pittsburgh, PA, USA).

Techniques: Inhibition, Activity Assay, Expressing, Western Blot, Software, Incubation, MTT Assay, Staining, Control, Phospho-proteomics

Journal: British Journal of Cancer

Article Title: Targeting PIM kinase enhances the activity of sunitinib in renal cell carcinoma

doi: 10.1038/bjc.2011.426

Figure Lengend Snippet: SGI-1776 reduces Bad phosphorylation and induces apoptosis in RCC tumours. ( A ) Phospho- and total Bad immunohistochemistry. Tumours were stained with either an antibody to phospho (Ser112) or total Bad and analysed by immunohistochemistry. The relative intensity of phospho-Bad expression was measured using Image-Pro Plus software Version 6.2.1. Mean±s.d., n =5. * Indicates a significant difference compared with controls. P <0.05. No significant differences were observed in total Bad expression in the different treatment groups. ( B ) SGI-1776 and sunitinib induce apoptosis. Apoptosis was measured by TUNEL staining as described in Materials and methods. Quantification was conducted by manually counting TUNEL-positive cells. Mean±s.d., n =5. * Indicates a significant difference compared with controls and ** represents a significant difference compared with single-agent treatments. P <0.05.

Article Snippet: Antibodies were obtained from the following commercial sources: anti-PIM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-Bad (Ser112) and Bad (Cell Signaling, Danvers, MA, USA); anti-actin and tubulin (Sigma-Aldrich, St Louis, MO, USA); anti-c-Myc, phospho-c-Myc (Ser62), and Bad (immunohistochemistry) (Novus Biologicals, Littleton, CO, USA); antiproliferating cell nuclear antigen (PCNA) (Dako, Glostrup, Denmark); goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratories, West Grove, PA, USA); Rat anti-mouse IgG2a-HRP (Serotec, Raleigh, NC, USA); and sheep anti-mouse-HRP and donkey anti-rabbit-HRP (Amersham, Pittsburgh, PA, USA).

Techniques: Phospho-proteomics, Immunohistochemistry, Staining, Expressing, Software, TUNEL Assay

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