Journal: BMC cancer

Article Title: Increased TCP11 gene expression can inhibit the proliferation, migration and promote apoptosis of cervical cancer cells.

doi: 10.1186/s12885-023-11129-1

Figure Lengend Snippet: Fig. 1 TCP11 gene is highly expressed in cervical cancer tissues and cells, and is closely associated with patients’ survival rate. A: The mRNA expression of TCP11 in normal cervical tissues and cervical cancer tissues were analyzed by GEPIA database. B: Immunohistochemical results of TCP11 protein expres sion in normal cervical tissues (n = 31) and cervical cancer tissues (n = 35) microarray. “-” indicates that the expression of TCP11 is negative; “+” indicates that the expression of TCP11 is weakly positive; “++” indicates that the expression of TCP11 is medium positive. Magnification: ×100 (top) and ×400 (bottom). C: Western blot was used to detect the expression of TCP11 protein in immortalized epithelial cells HaCaT and three cervical cancer cells. Full-length blots/ gels are presented in Supplementary Figure S1. D: qRT-PCR was used to detect the expression of TCP11 mRNA in immortalized epithelial cells HaCaT and three cervical cancer cells. E: GEPIA database was used to analyze the relationship between the expression of TCP11 and survival rate of cervical cancer patients. Data are presented as mean ± SD of at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: After incubation in the dark in 3% H2O2, they were incubated with TCP11 antibody (Proteintech, China, 1:200) overnight.

Techniques: Expressing, Immunohistochemical staining, Microarray, Western Blot, Quantitative RT-PCR

Journal: BMC cancer

Article Title: Increased TCP11 gene expression can inhibit the proliferation, migration and promote apoptosis of cervical cancer cells.

doi: 10.1186/s12885-023-11129-1

Figure Lengend Snippet: Fig. 2 TCP11 overexpression inhibits the proliferation of HeLa and SiHa cells. A: Western blot was used to detect the overexpression of TCP11 protein in HeLa and SiHa cells after lentivirus infection. Full-length blots/gels are presented in Supplementary Figure S2. B: qRT-PCR was used to detect the overex pression of TCP11 mRNA in HeLa and SiHa cells after lentivirus infection. C and D: The cell viability of HeLa and SiHa cells was detected by MTT and colony formation assays. E: The protein expression of Ki67 in HeLa and SiHa cells was detected by cellular immunohistochemistry. Magnification: ×100. The results were analyzed by Image Pro Plus software, and the mean density was calculated (mean density = IOD/area). F: Ki67 mRNA expression in HeLa and SiHa cells were detected by qRT-PCR. Data are presented as mean ± SD of at least 3 independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001

Article Snippet: After incubation in the dark in 3% H2O2, they were incubated with TCP11 antibody (Proteintech, China, 1:200) overnight.

Techniques: Over Expression, Western Blot, Infection, Quantitative RT-PCR, Expressing, Immunohistochemistry, Software

Journal: BMC cancer

Article Title: Increased TCP11 gene expression can inhibit the proliferation, migration and promote apoptosis of cervical cancer cells.

doi: 10.1186/s12885-023-11129-1

Figure Lengend Snippet: Fig. 3 TCP11 overexpression blocks cell cycle progression in HeLa and SiHa cells. A and B: Cell cycle distribution of HeLa and SiHa cells was determined by flow cytometry. C: Western blot was used to detect the protein expression of CDK1 and Cyclin B1 in HeLa and SiHa cells infected with overexpressing TCP11 lentivirus. Full-length blots/gels are presented in Supplementary Figure S3. D: qRT-PCR was used to detect the mRNA expression of CDK1 and Cyclin B1 in HeLa and SiHa cells infected with overexpressing TCP11 lentivirus. Data are presented as mean ± SD of at least 3 independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001

Article Snippet: After incubation in the dark in 3% H2O2, they were incubated with TCP11 antibody (Proteintech, China, 1:200) overnight.

Techniques: Over Expression, Flow Cytometry, Western Blot, Expressing, Infection, Quantitative RT-PCR

Journal: BMC cancer

Article Title: Increased TCP11 gene expression can inhibit the proliferation, migration and promote apoptosis of cervical cancer cells.

doi: 10.1186/s12885-023-11129-1

Figure Lengend Snippet: Fig. 4 TCP11 overexpression induces the apoptosis of HeLa and SiHa cells. A and B: Apoptosis of HeLa and SiHa cells were detected by flow cytometry. C: Western blot was used to detect the protein expression of caspase-3, cleaved-caspase-3 and cleaved-PARP in HeLa and SiHa cells infected with over expressing TCP11 lentivirus. Full-length blots/gels are presented in Supplementary Figure S4. Data are presented as mean ± SD of at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: After incubation in the dark in 3% H2O2, they were incubated with TCP11 antibody (Proteintech, China, 1:200) overnight.

Techniques: Over Expression, Flow Cytometry, Western Blot, Expressing, Infection

Journal: BMC cancer

Article Title: Increased TCP11 gene expression can inhibit the proliferation, migration and promote apoptosis of cervical cancer cells.

doi: 10.1186/s12885-023-11129-1

Figure Lengend Snippet: Fig. 5 TCP11 overexpression inhibits cervical cancer cell migration by inhibiting EMT of HeLa and SiHa cells. A and B: Cell scratch assay was used to detect the migration ability of HeLa and SiHa cells. C and D: Transwell migration assay was used to detect the migration ability of HeLa and SiHa cells. E: Western blot was used to detect the protein expression of EMT-related molecules ZO-1, E-cadherin, Claudin-1, Snail, Vimentin and β-catenin in HeLa and SiHa cells infected with overexpressing TCP11 lentivirus. Full-length blots/gels are presented in Supplementary Figure S5. F: qRT-PCR was used to detect the mRNA expression of EMT-related molecules ZO-1 and E-cadherin in HeLa and SiHa cells infected with overexpressing TCP11 lentivirus. Data are presented as mean ± SD of at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: After incubation in the dark in 3% H2O2, they were incubated with TCP11 antibody (Proteintech, China, 1:200) overnight.

Techniques: Over Expression, Migration, Wound Healing Assay, Transwell Migration Assay, Western Blot, Expressing, Infection, Quantitative RT-PCR

Journal: BMC cancer

Article Title: Increased TCP11 gene expression can inhibit the proliferation, migration and promote apoptosis of cervical cancer cells.

doi: 10.1186/s12885-023-11129-1

Figure Lengend Snippet: Fig. 6 TCP11 knockdown promotes the proliferation of HeLa and SiHa cells and the migration of HeLa cells. A: Western blot was used to detect the knock down effect of three designed and constructed siRNA fragments on TCP11 in HeLa and SiHa cells. Full-length blots/gels are presented in Supplementary Figure S6. B: qRT-PCR was used to detect the knockdown effect of three siRNA fragments on TCP11 in HeLa and SiHa cells. C: MTT assay was used to detect the effect of TCP11 knockdown on the proliferation of HeLa and SiHa cells. D: Colony formation assay was used to detect the effect of TCP11 knockdown on the proliferation of HeLa cells. E: Transwell migration assay was used to detect the effect of TCP11 knockdown on the migration of HeLa cells. Data are presented as mean ± SD of at least 3 independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001

Article Snippet: After incubation in the dark in 3% H2O2, they were incubated with TCP11 antibody (Proteintech, China, 1:200) overnight.

Techniques: Knockdown, Migration, Western Blot, Construct, Quantitative RT-PCR, MTT Assay, Colony Assay, Transwell Migration Assay

Journal: Cell Reports Methods

Article Title: TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy

doi: 10.1016/j.crmeth.2022.100311

Figure Lengend Snippet: TissUExM is a versatile method for various models and organelles (A) 110 h post-fertilization (hpf) myosin II regulatory light chain-GFP Drosophila wing disc, stained for GFP and a-tub; DAPI is in blue. Whole wing (10×), apical side of the pouch with dividing cells (10×), microtubule cytoskeleton (63×), and deconvolved centrosome with a pair of centrioles in top and side views (63×). Scale bars: 100, 5, 1, and 100 nm. (B) Centrioles in top view. Mean ± SD tubulin roundness: 0.929 ± 0.042. n = 17 centrioles from three independent experiments. Mean ± SD tubulin diameter: 175 ± 9 nm. n = 17 centrioles from three independent experiments. (C) E8.5 whole mouse, stained for PolyE and aTub. DAPI is in blue. Top left: whole embryo (10×); bottom left: node enriched in cilia (10×); top right: maximum projection of a node cilium with inset on the tip (63×); bottom right: maximum projection and deconvolution applied to two cilia with magnification of the cilium base (63×). Right panel shows a BB/cilium complex architecture with subregions such as the TZ and the cilium tip. Scale bars: 100, 1, and 5μm and 200 nm. (D) Centrioles and cilia in top view. Mean ± SD tubulin roundness: 0.932 ± 0.035. n = 22 centrioles from three independent experiments. Mean ± SD tubulin diameter: 234 ± 13 nm. n = 22 centrioles from three independent experiments. Mean ± SD ciliary PolyE length in neural tube: 1,075 ± 411 nm, somite: 899 ± 385 nm, and node 2,956 ± 1,384 nm. n ≥ 20 cilia/tissue from three independent experiments. One-way ANOVA and Kruskal-Wallis ∗∗∗∗p < 0.0001. Mean ± SD ciliary PolyE width in neural tube: 79 ± 33 nm, somite: 93 ± 30 nm, and node: 215 ± 37 nm. n ≥ 20 cilia/tissue from three independent experiments. One-way ANOVA and Kruskal-Wallis ∗∗∗∗p < 0.0001. (E) Organelles in E8.5 mouse. Mitochondria (TOMM20), vesicles (clathrin heavy chain) and tight junctions (ZO1), Golgi apparatus (GM130), nuclear envelope (Lamin B1), and nuclear pore complexes (NUP205). 63×/1.20, Scale bars: 5 and 1 μm. Insets show the regions for fluorescence intensity profiles. Note that no deconvolution was applied. See also Figure S3 .

Article Snippet: NUP205, Rabbit poly. , Proteintech , Cat#24439-1-AP, Ag19832; RRID: AB_2879550.

Techniques: Staining, Fluorescence

Journal: Cell Reports Methods

Article Title: TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy

doi: 10.1016/j.crmeth.2022.100311

Figure Lengend Snippet:

Article Snippet: NUP205, Rabbit poly. , Proteintech , Cat#24439-1-AP, Ag19832; RRID: AB_2879550.

Techniques: Recombinant, Saline, Software

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: Homozygous Myo18a deletion is lethal. A and B, schematic diagram of the Myo18a knockout first targeting strategy and conversion of the tm1a (knockout first) allele to tm1c (floxed) and tm1d (knockout) alleles, respectively. Germ line deletion of Myo18a was produced by interbreeding Myo18atm1c and PGK-Cre mice (to yield Myo18atm1d). Myo18a targeting was confirmed by Southern blot analysis. C, PCR genotyping of offspring derived from crossing HET knockout first (Myo18a+/tm1a × Myo18a+/tm1a) mice. Notably, no mice HOM for the knockout first allele were detected. D, PCR genotyping of offspring derived from crossing heterozygous floxed (Myo18a+/tm1c × Myo18a+/tm1c) or heterozygous Myo18a knockout (Myo18a+/tm1d × Myo18a+/tm1d) mice. E, no homozygous Myo18a knockout (Myo18atm1d/tm1d) mice were detected at E13.5, indicating that Myo18a deletion is embryonic lethal.

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Knock-Out, Produced, Southern Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: X-gal staining and histology of Myo18atm1b embryos. A, conversion of the tm1a (knockout first) allele to the tm1b (reporter knockout) allele by crossing heterozygous knockout first (Myo18a+/tm1a) mice with ACTB-Cre mice. B, timing of embryonic lethality by timed intercrossings of heterozygous knockout reporter (Myo18a+/tm1b) mice. No viable homozygous embryos were detected after E12.5. *, data from crossing Myo18a+/tm1a × Myo18a+/tm1a. C, whole-mount X-gal staining of HET Myo18a reporter knockout embryos at E11.5 and E10.5. Scale bars, 1 mm. D, histological sections of the heart obtained from X-gal–stained WT and HOM Myo18a reporter knockout E10.5 embryos. Arrows, X-gal staining of myocardial cells. Scale bars, 250 μm. AVC, atrioventricular cushions; V, ventricle; A, atrium. E, hematoxylin and eosin (HE) staining of sagittal sections of E10.5 HOM knockout (Myo18atm1b/tm1b) and WT embryos. OFT, outflow tract; AVC, atrioventricular cushions; V, ventricle; A, atrium.

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Staining, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: Mouse ventricular myocytes express a novel isoform of Myo18A and cardiac-restricted Myo18a deletion is lethal. A, generation of cardiac-restricted Myo18a knockout mice. Mice harboring the floxed Myo18a allele (Myo18atm1c) were crossed with Tnnt2-Cre mice, in which Cre recombinase is expressed in cardiac myocytes. Conditional knockout mice were further crossed with Cre reporter (R26R-EYFP) mice to derive triple mutant (cardiac-restricted Myo18a knockout with Cre reporter) mice. B, bright field and fluorescence images of ventricular myocytes isolated from a Myo18a+/tm1c/Tnnt2-Cre/R26R-EYFP mouse. Green (EYFP) fluorescence is an indicator of Cre recombinase activity. Scale bar, 10 μm. C, no homozygous cardiac-restricted Myo18a knockout (Myo18atm1c/tm1c/Tnnt2-Cre) mice were obtained after crossing Myo18atm1c/tm1c and Myo18a+/tm1c/Tnnt2-Cre mice, indicating that conditional deletion of Myo18a in cardiac myocytes is lethal. No Myo18atm1c/tm1c/Tnnt2-Cre embryos were detected at E13.5, and three dead embryos were detected at E14.5. D, photographs of HET and homozygous cardiac-restricted Myo18a knockout embryos at day E14.5. Scale bar, 0.5 mm. E, Western blotting analyses. A custom antibody against the last 18 amino acids of mouse Myo18A (anti-Myo18A-C-term) recognizes the Myo18A isoforms Myo18Aα and Myo18Aβ. Lysates were prepared from HeLa cells transiently expressing EGFP-Myo18Aα (mouse) or EGFP-Myo18Aβ (mouse). The custom anti-Myo18A-C-term antibody did not detect Myo18A in mouse heart lysate, whereas an antibody against the coiled-coil (CC) domain of Myo18A detected a larger than expected band on the same membrane.

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Knock-Out, Mutagenesis, Fluorescence, Isolation, Activity Assay, Western Blot, Expressing, Membrane

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: Cloning and sequencing of the novel isoform of Myo18A expressed in mouse ventricular myocytes. A, representative Sashimi plot of data obtained by mouse ventricular myocyte RNA-Seq (n = 6 mice). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles (with dashed lines) indicate exons that are abundant in the ventricular myocyte transcript but absent in the aligned annotated genomic references corresponding to transcripts for Myo18Aα and Myo18Aβ. B, domain structure of the novel cardiac isoform of Myo18A, Myo18Aγ, as well as, for comparison, the domain structures of Myo18Aα and Myo18Aβ. The recognition sites of various anti-Myo18A antibodies used in this study are indicated below. Notably, the custom (anti-Myo18A-C-term) antibody is unable to recognize the novel C terminus of Myo18Aγ. PPII, polyproline II helix; KE, KE-rich region; PDZ, PDZ domain; IQ, IQ motif. C, alignment of the N-terminal protein sequence of Myo18Aγ, derived from cloning and sequencing cDNA from mouse ventricular myocytes, with a mouse protein sequence of 219 residues from the Ensembl database (MGP_FVBNJ_P0030691).

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Cloning, Sequencing, RNA Sequencing, Comparison, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: Human homolog of mouse Myo18Aγ and mouse–human sequence alignment. A, representative Sashimi plot of data obtained by RNA-Seq analysis of human atrial tissue (n = 10 subjects). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles indicate exons that are detected in the human atrial tissue transcript but absent in the aligned genomic references corresponding to transcripts (a–f) for human MYO18A. The accession numbers are as follows: MYO18Aa (NM_078471), MYO18Ab (NM_203318), MYO18Ac (NM_001346765), MYO18Ad (NM_001346766), MYO18Ae (NM_001346767), and MYO18Af (NM_001346768). B, sequence alignment of the N and C termini (N- and C-term) of mouse (Mus musculus (Mus-mus)) Myo18Aγ with human (Homo sapiens (Hom-sap)) atrial MYO18A. Identical residues are highlighted in gray and indicated by an asterisk below the alignment. Conserved mutations (residues replaced by another amino acid with similar biochemical properties) are indicated by a colon, and semiconserved mutations are denoted by a period. The red numbers below the sequences for Homo sapiens correspond to the (red) numbered exons depicted in the Sashimi plot above.

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Sequencing, RNA Sequencing

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: Subcellular localization of Myo18Aγ in neonatal rat ventricular cardiomyocytes. A, NRVMs were lipofected with expression plasmids encoding EGFP-tagged mouse Myo18Aγ (Myo18Aγ-EGFP; middle row) and mScarlet-I-tagged mouse Cypher 1c (middle column) or mScarlet-I–tagged rat Myl2 (right column). Alternatively, cells fixed 4 days post-transfection were labeled with antibodies directed against sarcomeric α-actinin (left column). Images were obtained by laser-scanning confocal microscopy. Scale bars, 10 μm. B, enlarged views of the rectangular regions of interest shown in the top row of A. A horizontal line was drawn across these regions of interest to obtain the corresponding density plots, shown below. a.u., arbitrary units.

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Expressing, Transfection, Labeling, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: Ultrastructural analysis of Myo18a-deficient E10.5 embryo hearts. A and B, high-magnification images, obtained by transmission EM, showing well-defined sarcomeres in WT and HET Myo18a knockout (Myo18a+/tm1b) E10.5 embryo hearts. The insets indicate parallel bundled filaments. C, highly disorganized sarcomeres in HOM Myo18a knockout (Myo18atm1b/tm1b) E10.5 embryo hearts. Z, Z-disc; I, I-band; A, A-band. Scale bars, 250 nm.

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Transmission Assay, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: Myo18a and Myo18b are the predominantly expressed unconventional myosins in mouse ventricular myocytes, and Myo18Aγ is detected in both mouse cardiac and skeletal muscle. A, expression levels of unconventional myosins in mouse ventricular myocytes. Data were obtained by RNA sequence analysis (n = 6 mice), and expression levels are indexed as FPKM (fragments per kilobase of transcript per million mapped reads). B, expression levels of conventional (class II) myosins in mouse ventricular myocytes. Note that Myh6, the dominant cardiac-specific myosin heavy chain isoform in adult mice, is highly expressed (2 of the 6 samples exceeded the detection range; not shown). C, Western blot analysis. Anti-Myo18A coiled-coil domain antibodies were used to detect Myo18A isoforms expressed in HeLa cells, mouse heart, and mouse skeletal (gastrocnemius) muscle. Error bars, S.E.

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Expressing, Sequencing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: Generation of myeloid-restricted Myo18a knockout mice and expression of the splice variants Myo18Aα and Myo18Aβ in macrophages. A, triple mutant Myo18a cKO mice. Shown is a schematic diagram of the floxed Myo18a allele (Myo18atm1c) and conversion to a myeloid-restricted Myo18a knockout allele with a fluorescent reporter (R26R-EYFP) of Cre recombinase activity. The bright field and fluorescence images on the right, obtained by spinning disk confocal microscopy, show resident peritoneal macrophages isolated from a Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mouse. Images are 120 × 120 μm. B, Western blot analysis of purified WT (F4/80+ cells) versus conditional Myo18a knockout macrophages (EYFP+ cells). In each lane, lysate from 2 × 105 cells was loaded. C, Western blot analysis of Myo18A protein detected in the following cell lysates: untransfected (human) HeLa cells, HeLa cells transfected with mouse EGFP-Myo18Aβ1, HeLa cells transfected with mouse EGFP-Myo18Aβ2, 1.5 × 105 purified peritoneal F4/80+ cells (macrophages) from WT mice, 1.5 × 105 purified peritoneal EYFP+ (Cre recombinase–positive) cells from Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mice, and (mouse) NIH-3T3 cells. A separate blot shows the relative intensities of Myo18A alternative splicing isoforms. D, relative protein levels of Myo18Aα (α) and Myo18Aβ (β) in purified mouse resident macrophages. Error bars, S.E. E, migration plots of WT and Myo18a-deficient macrophages in a complement C5a gradient. F, summary plots of chemotactic efficiency (chemotaxis index) and mean velocity (n = 100 cells (4 mice) in each group); n.s. = not significant at the 0.05 level of significance (Mann–Whitney U test). Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe) was used for the above Western blotting analyses.

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Knock-Out, Expressing, Mutagenesis, Activity Assay, Fluorescence, Confocal Microscopy, Isolation, Western Blot, Purification, Transfection, Alternative Splicing, Migration, Chemotaxis Assay, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

doi: 10.1074/jbc.RA118.004560

Figure Lengend Snippet: Myo18A does not localize to Golgi and Myo18a-deficient macrophages have normal Golgi morphology. A, resolution of the Zeiss Elyra superresolution structured illumination microscope (SR-SIM) determined by point spread function measurements. Subdiffraction-sized fluorescent beads (100 nm diameter) were imaged via a ×63 oil immersion objective lens (numerical aperture 1.46), and stacks were obtained at 110-nm steps. B, SR-SIM images of fixed WT and Myo18a-deficient (Myo18atm1c/tm1c/LysM-Cre; cKO) macrophages labeled with anti-GM130 (cis-Golgi) or anti-Giantin (Golgi membrane) antibodies (green) and the nucleic acid stain 4′,6-diamidino-2-phenylindole (blue). C, summary plots of Golgi area and Golgi extent (the fraction of the nuclear perimeter overlapped by Golgi cisternae) in WT versus Myo18a-deficient macrophages; n.s., not significant at the 0.05 level of significance (Mann–Whitney U test). D, SR-SIM images of living HeLa cells stably transfected with Myo18A-EGFP (green) and transiently transfected with the Golgi probe RFP-Golgi (red). Right, box plot of Pearson's correlation coefficient. Scale bar, 10 μm.

Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

Techniques: Microscopy, Labeling, Membrane, Staining, MANN-WHITNEY, Stable Transfection, Transfection

Journal: Frontiers in Immunology

Article Title: Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis

doi: 10.3389/fimmu.2018.01197

Figure Lengend Snippet: Primer sequences for real-time PCR.

Article Snippet: The sections were then incubated with rabbit antibodies against human NLRP3 (#19771-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRP1 (#12256-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRC4 (#A7382 at a 1:50 dilution; AB Clonal Inc., USA) and AIM2 (#20590-1-AP at a 1:100 dilution; Proteintech Group Inc., USA) in a humidity box at 4°C overnight.

Techniques:

Journal: Frontiers in Immunology

Article Title: Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis

doi: 10.3389/fimmu.2018.01197

Figure Lengend Snippet: Increased mRNA and protein expression of inflammasome components in thyroid tissues from AIT patients. (A) The mRNA expression levels of NLRP1, NLRP3, NLRC4, absent in melanoma 2 (AIM2), ASC, and caspase-1 in thyroid tissues from AIT patients and controls ( N = 50 in each group). Representative stripe images (B) and corresponding quantitative analysis (C) of the NLRP1, NLRP3, NLRC4, AIM2, ASC, caspase-1 and caspase-1 p20 protein levels in thyroid tissues from AIT patients and controls ( N = 20 in each group). The relative mRNA and protein expression levels were corrected to GAPDH expression. The columns represent the mean ± SD values. Student’s t -test or Mann–Whitney U -test was used for comparison between the AIT and control groups. * P < 0.05, ** P < 0.01. AIT, autoimmune thyroiditis; CON, controls.

Article Snippet: The sections were then incubated with rabbit antibodies against human NLRP3 (#19771-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRP1 (#12256-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRC4 (#A7382 at a 1:50 dilution; AB Clonal Inc., USA) and AIM2 (#20590-1-AP at a 1:100 dilution; Proteintech Group Inc., USA) in a humidity box at 4°C overnight.

Techniques: Expressing, MANN-WHITNEY, Comparison, Control

Journal: Frontiers in Immunology

Article Title: Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis

doi: 10.3389/fimmu.2018.01197

Figure Lengend Snippet: Localization of enhanced expression of NLRP1, NLRP3, NLRC4, and absent in melanoma 2 (AIM2) in thyroid cells in areas of lymphatic infiltration. (A) Representative immunohistochemical staining images of AIM2, NLRP3, NLRP1, and NLRC4 in thyroid tissue sections from AIT patients and controls (400× magnification, light microscope). (B) Quantified expression levels of AIM2, NLRP3, NLRP1, and NLRC4 in control and AIT tissues ( N = 20 in each group). The columns represent the mean ± SD values. Three views were randomly selected in each subject. Statistical analysis was carried out using Student’s t -test. * P < 0.05, ** P < 0.01. AIT, autoimmune thyroiditis; CON, controls; IIA, inflammatory infiltration area; NIIA, non-inflammatory infiltration area.

Article Snippet: The sections were then incubated with rabbit antibodies against human NLRP3 (#19771-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRP1 (#12256-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRC4 (#A7382 at a 1:50 dilution; AB Clonal Inc., USA) and AIM2 (#20590-1-AP at a 1:100 dilution; Proteintech Group Inc., USA) in a humidity box at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining, Light Microscopy, Control

Journal: Frontiers in Immunology

Article Title: Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis

doi: 10.3389/fimmu.2018.01197

Figure Lengend Snippet: Correlation between the mRNA levels of inflammasome components in thyroid tissues and autoantibody levels in the serum in autoimmune thyroiditis (AIT) patients. Correlation between the NLRP1/ASC mRNA levels in thyroid tissue and serum thyroid peroxidase antibody (TPOAb)/thyroglobulin antibody (TgAb) levels in the AIT group (A–C) . N = 50 in each group. A bivariate correlation analysis was performed using the Spearman rank test. R represents the Spearman correlation coefficient.

Article Snippet: The sections were then incubated with rabbit antibodies against human NLRP3 (#19771-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRP1 (#12256-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRC4 (#A7382 at a 1:50 dilution; AB Clonal Inc., USA) and AIM2 (#20590-1-AP at a 1:100 dilution; Proteintech Group Inc., USA) in a humidity box at 4°C overnight.

Techniques:

Journal: Frontiers in Immunology

Article Title: Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis

doi: 10.3389/fimmu.2018.01197

Figure Lengend Snippet: Correlation between NLRP3, NLRP1, NLRC4, absent in melanoma 2 (AIM2), ASC, caspase-1, IL-1β, and IL-18 expression in the autoimmune thyroiditis group.

Article Snippet: The sections were then incubated with rabbit antibodies against human NLRP3 (#19771-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRP1 (#12256-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRC4 (#A7382 at a 1:50 dilution; AB Clonal Inc., USA) and AIM2 (#20590-1-AP at a 1:100 dilution; Proteintech Group Inc., USA) in a humidity box at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis

doi: 10.3389/fimmu.2018.01197

Figure Lengend Snippet: Correlation between NLRP3, NLRP1, NLRC4, absent in melanoma 2 (AIM2), ASC, caspase-1, IL-1β, and IL-18 expression in the CON group.

Article Snippet: The sections were then incubated with rabbit antibodies against human NLRP3 (#19771-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRP1 (#12256-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRC4 (#A7382 at a 1:50 dilution; AB Clonal Inc., USA) and AIM2 (#20590-1-AP at a 1:100 dilution; Proteintech Group Inc., USA) in a humidity box at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis

doi: 10.3389/fimmu.2018.01197

Figure Lengend Snippet: Increased expression of inflammasome components in thyroid cells on stimulation with IFN-γ and tumor necrosis factor-α (TNF-α). (A) Changes in NLRP3, absent in melanoma 2 (AIM2), NLRP1, NLRC4, ASC, and caspase-1 protein expression detected by western blot after incubation with 500 IU/ml IFN-γ or 500 IU/ml TNF-α for 24 h. Changes in NLRP3, AIM2, NLRP1, NLRC4, ASC, Caspase-1, IL-1β, and IL-18 mRNA expression detected by real-time PCR after incubation with (B) gradient IFN-γ (250, 500, and 1,000 IU/ml) for 24 h or (C) gradient TNF-α (125, 250, and 500 IU/ml) for 24 h. The relative mRNA and protein expression levels were corrected to those of untreated controls. (D) IL-18 and IL-1β levels in cell supernatant, as determined by enzyme-linked immunosorbent assay, after stimulation with 500 IU/ml IFN-γ, 500 IU/ml TNF-α or a combination of the two for 24 h. The columns represent the mean ± SD values. Data were obtained from three independent experiments. One-way ANOVA followed by Bonferroni correction was used for paired comparisons. * P < 0.05, ** P < 0.01.

Article Snippet: The sections were then incubated with rabbit antibodies against human NLRP3 (#19771-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRP1 (#12256-1-AP at a 1:100 dilution; Proteintech Group Inc., USA), NLRC4 (#A7382 at a 1:50 dilution; AB Clonal Inc., USA) and AIM2 (#20590-1-AP at a 1:100 dilution; Proteintech Group Inc., USA) in a humidity box at 4°C overnight.

Techniques: Expressing, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay