Journal: Oncology letters

Article Title: Reversal of the tamoxifen‑resistant breast cancer malignant phenotype by proliferation inhibition with bromosulfonamidine amino‑podophyllotoxin.

doi: 10.3892/ol.2024.14506

Figure Lengend Snippet: Figure 4. Effect of BSAPPT on apoptosis and cycle‑related gene or protein expression in MCF‑7, MCF7/TAMR and other cancer cells. (A) mRNA expres‑ sion levels of genes linked to the cell cycle and apoptosis were measured by qPCR both before and after MCF‑7 and MCF7/TAMR cells were treated with 10 µg/ml BSAPPT. (B) Western blotting detection of apoptotic and cycle‑related protein expression variations in MCF‑7 and MCF7/TAMR cells before and after using 10 µg/ml BSAPPT. Results of qPCR analysis that assessed differences in the level of expression of genes linked to the cell cycle and apoptosis before and after (C) A549 and (D) MDA‑MB‑231 cells were treated with 10 µg/ml BSAPPT. *P<0.05; **P<0.01; ***P<0.001. BSAPPT, bromosulfonamidine amino‑podophyllotoxin; qPCR, quantitative PCR; Bcl‑2, B‑cell lymphoma 2; Caspase, cysteine aspartic acid‑specific protease; PLK, polo like kinase; CCNB1, cyclin B1; TPX2, targeting protein for Xklp2; Bax, Bcl‑2 associated X; Cyt‑C, cytochrome c; Apaf‑1, apoptotic protease activating factor 1.

Article Snippet: Mouse anti‐human Caspase‐9 antibodies (cat. no. 9508S; 1:1,000) were purchased from Cell Signaling Technology, Inc., rabbit anti‐human Bcl‐2 (cat. no. BA0412; 1:1,000) and cyclin B1 (CCNB1; cat. no. BA0766; 1:1,000) antibodies were purchased from Wuhan Boster Biological Technology, Ltd., rabbit anti‐human polo like kinase (PLK)‐1 antibodies (cat. no. 10305‐1‐AP; 1:1,000) and targeting protein for Xklp2 (TPX2) antibodies (cat. no. 11741‐1‐AP; 1:2,000) were purchased from Proteintech Group, Inc., and the rabbit anti‐human PLK‐4 antibodies (cat. no. A9863; 1:1,000) were purchased from ABclonal Biotech Co., Ltd.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo

doi: 10.3389/fphar.2018.01045

Figure Lengend Snippet: The expression levels of MYCT1 in BM of AML patients and Healthy controls. Relative mRNA (A) and protein (B) levels of MYCT1 in the BM of AML patients and healthy controls were tested by RT-PCR and Western blot analysis, respectively ( n = 50). (C) Methylation density of MYCT1 gene in AML patients and Healthy controls as analyzed by BSP ( n = 9); (D) Methylation status of the specific promoter region of the MYCT1 gene. Each line of circles indicated the sequence of an individual clone; ∘ represented an unmethylated CpG site and ∙ represented a methylated CpG site. ∗∗ P < 0.01 versus healthy group and BM, bone marrow.

Article Snippet: Anti-MYCT1 antibody (dilution rate 1:1000) was purchased from Proteintech Group Inc. (Wuhan, Hubei, China), anti-cyclin D1 (dilution rate 1:500) antibody was purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, United States), anti-cyclin E (dilution rate 1:1000) and anti-cleaved caspase-3 (dilution rate 1:1000) antibodies were purchased from Abcam (Cambridge, MA, United States), anti-B-cell lymphoma 2 (anti-Bcl-2, dilution rate 1:1000) and anti-Bcl-2-associated X protein (anti-Bax, dilution rate 1:1000) antibodies were purchased from Sangon Biotech (Shanghai, China), anti-cleaved caspase-9 antibody (dilution rate 1:1000) was purchased from CST Inc. (Danvers, MA, United States) and anti-GAPDH antibody (dilution rate 1:500) was purchased from Bioss Inc. (Woburn, MA, United States).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Methylation, Sequencing

Journal: Frontiers in Pharmacology

Article Title: Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo

doi: 10.3389/fphar.2018.01045

Figure Lengend Snippet: Correlation between MYCT1 expression and clinical characteristics of AML patients ( n = 50).

Article Snippet: Anti-MYCT1 antibody (dilution rate 1:1000) was purchased from Proteintech Group Inc. (Wuhan, Hubei, China), anti-cyclin D1 (dilution rate 1:500) antibody was purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, United States), anti-cyclin E (dilution rate 1:1000) and anti-cleaved caspase-3 (dilution rate 1:1000) antibodies were purchased from Abcam (Cambridge, MA, United States), anti-B-cell lymphoma 2 (anti-Bcl-2, dilution rate 1:1000) and anti-Bcl-2-associated X protein (anti-Bax, dilution rate 1:1000) antibodies were purchased from Sangon Biotech (Shanghai, China), anti-cleaved caspase-9 antibody (dilution rate 1:1000) was purchased from CST Inc. (Danvers, MA, United States) and anti-GAPDH antibody (dilution rate 1:500) was purchased from Bioss Inc. (Woburn, MA, United States).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo

doi: 10.3389/fphar.2018.01045

Figure Lengend Snippet: Overexpression of MYCT1 in HL-60 and KG-1a AML cells by lentiviral infection. The mRNA (A) and protein (B) levels of MYCT1 in HL-60 and KG-1a cell lines were examined by RT-PCR and Western blot analysis, respectively. (C–F) HL-60 and KG-1a cells were infected with negative control lentiviral particles (Lv-NC) or lentiviral particles overexpressing MYCT1 (Lv-MYCT1). The mRNA (C,D) and protein (E,F) levels of MYCT1 in HL-60 (C,E) and KG-1a (D,F) cells were assessed by RT-PCR and Western blot analysis, respectively. ∗∗ P < 0.01 versus HL-Lv-NC or KG-Lv-NC cells.

Article Snippet: Anti-MYCT1 antibody (dilution rate 1:1000) was purchased from Proteintech Group Inc. (Wuhan, Hubei, China), anti-cyclin D1 (dilution rate 1:500) antibody was purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, United States), anti-cyclin E (dilution rate 1:1000) and anti-cleaved caspase-3 (dilution rate 1:1000) antibodies were purchased from Abcam (Cambridge, MA, United States), anti-B-cell lymphoma 2 (anti-Bcl-2, dilution rate 1:1000) and anti-Bcl-2-associated X protein (anti-Bax, dilution rate 1:1000) antibodies were purchased from Sangon Biotech (Shanghai, China), anti-cleaved caspase-9 antibody (dilution rate 1:1000) was purchased from CST Inc. (Danvers, MA, United States) and anti-GAPDH antibody (dilution rate 1:500) was purchased from Bioss Inc. (Woburn, MA, United States).

Techniques: Over Expression, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo

doi: 10.3389/fphar.2018.01045

Figure Lengend Snippet: Overexpression of MYCT1 inhibits cell proliferation and induces cell cycle arrest in HL-60 and KG-1a cells. Cells proliferation was measured by CCK-8 assay in HL-60 (A) and KG-1a (B) cells at 24 h, 48 h, 72 h and 96 h after lentiviral delivery. (C–F) MYCT1 overexpression-induced cell cycle arrest in HL-60 and KG-1a cells. Changes in cell cycle distribution of MYCT1-overexpressing HL-60 (C) and KG-1a (D) cells were determined by FACS analysis, and the proportions of cells in G 0 /G 1 , S and G 2 /M phase were calculated (D,F) . (G–J) The expression levels of cell cycle regulatory proteins in HL-60 (G,H) and KG-1a (I,J) cells were examined by Western blot analysis. ∗ P < 0.05 and ∗∗ P < 0.01 versus HL-Lv-NC or KG-Lv-NC cells.

Article Snippet: Anti-MYCT1 antibody (dilution rate 1:1000) was purchased from Proteintech Group Inc. (Wuhan, Hubei, China), anti-cyclin D1 (dilution rate 1:500) antibody was purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, United States), anti-cyclin E (dilution rate 1:1000) and anti-cleaved caspase-3 (dilution rate 1:1000) antibodies were purchased from Abcam (Cambridge, MA, United States), anti-B-cell lymphoma 2 (anti-Bcl-2, dilution rate 1:1000) and anti-Bcl-2-associated X protein (anti-Bax, dilution rate 1:1000) antibodies were purchased from Sangon Biotech (Shanghai, China), anti-cleaved caspase-9 antibody (dilution rate 1:1000) was purchased from CST Inc. (Danvers, MA, United States) and anti-GAPDH antibody (dilution rate 1:500) was purchased from Bioss Inc. (Woburn, MA, United States).

Techniques: Over Expression, CCK-8 Assay, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo

doi: 10.3389/fphar.2018.01045

Figure Lengend Snippet: Overexpression of MYCT1 induces apoptosis in HL-60 and KG-1a cells. (A,B) The apoptotic cells were detected by FACS analysis after staining with PI/Annexin V-Light 650, and the apoptosis rates were shown in parts (C,D) (apoptotic cell fraction = UR + LR). UR quadrant represents Annexin V and PI positive staining cells (apoptotic cells), while LR quadrant represents Annexin V positive and PI negative staining cells (early apoptotic cells). (E,F) Apoptosis, as induced by overexpression of MYCT1 in HL-60 (E) and KG-1a (F) cells, was determined by Hoechst staining. ∗∗ P < 0.01 versus HL-Lv-NC or KG-Lv-NC cells. Scale bar: 50 μm. (G,H) The levels of apoptosis-related proteins, including cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax were assessed by Western blot analysis. (I,J) Quantitative analysis of the gray intensity values. ∗∗ P < 0.01 versus HL-Lv-NC or KG-Lv-NC cells. UR, Upper right and LR, Lower right.

Article Snippet: Anti-MYCT1 antibody (dilution rate 1:1000) was purchased from Proteintech Group Inc. (Wuhan, Hubei, China), anti-cyclin D1 (dilution rate 1:500) antibody was purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, United States), anti-cyclin E (dilution rate 1:1000) and anti-cleaved caspase-3 (dilution rate 1:1000) antibodies were purchased from Abcam (Cambridge, MA, United States), anti-B-cell lymphoma 2 (anti-Bcl-2, dilution rate 1:1000) and anti-Bcl-2-associated X protein (anti-Bax, dilution rate 1:1000) antibodies were purchased from Sangon Biotech (Shanghai, China), anti-cleaved caspase-9 antibody (dilution rate 1:1000) was purchased from CST Inc. (Danvers, MA, United States) and anti-GAPDH antibody (dilution rate 1:500) was purchased from Bioss Inc. (Woburn, MA, United States).

Techniques: Over Expression, Staining, Negative Staining, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo

doi: 10.3389/fphar.2018.01045

Figure Lengend Snippet: Overexpression of MYCT1 delays the growth of AML xenograft tumors in vivo . HL-60 and KG-1a cells were first subcutaneously implanted into the flanks of BALB/c-nu mice. Mice were given intratumoral injections of Lv-NC or Lv-MYCT1 at day 7, 12, and 17. (A,B) Tumor volumes of AML xenografts in different groups. (C,D) Photographs of the mice bearing AML xenograft tumors (C,D , upper panel ) and the isolated tumors (C,D , lower panel ) . (E,F) Tumor weights of AML xenografts in different groups. ∗ P < 0.05 and ∗∗ P < 0.01 versus HL-Lv-NC or KG-Lv-NC cells.

Article Snippet: Anti-MYCT1 antibody (dilution rate 1:1000) was purchased from Proteintech Group Inc. (Wuhan, Hubei, China), anti-cyclin D1 (dilution rate 1:500) antibody was purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, United States), anti-cyclin E (dilution rate 1:1000) and anti-cleaved caspase-3 (dilution rate 1:1000) antibodies were purchased from Abcam (Cambridge, MA, United States), anti-B-cell lymphoma 2 (anti-Bcl-2, dilution rate 1:1000) and anti-Bcl-2-associated X protein (anti-Bax, dilution rate 1:1000) antibodies were purchased from Sangon Biotech (Shanghai, China), anti-cleaved caspase-9 antibody (dilution rate 1:1000) was purchased from CST Inc. (Danvers, MA, United States) and anti-GAPDH antibody (dilution rate 1:500) was purchased from Bioss Inc. (Woburn, MA, United States).

Techniques: Over Expression, In Vivo, Isolation

Journal: Frontiers in Pharmacology

Article Title: Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo

doi: 10.3389/fphar.2018.01045

Figure Lengend Snippet: Overexpression of MYCT1 induces tumor-cell apoptosis in a mouse xenograft model in vivo . (A–D) The expression levels of MYCT1 in AML xenograft tumors determined by Western blot analysis. (E,F) Histological examination of xenograft tumors in different groups by HE staining. Scale bar: 50 μm. (G,H) Apoptosis in the xenograft tumors was determined by TUNEL assay. Scale bar: 50 μm and ∗∗ P < 0.01 versus HL-Lv-NC or KG-Lv-NC cells.

Article Snippet: Anti-MYCT1 antibody (dilution rate 1:1000) was purchased from Proteintech Group Inc. (Wuhan, Hubei, China), anti-cyclin D1 (dilution rate 1:500) antibody was purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, United States), anti-cyclin E (dilution rate 1:1000) and anti-cleaved caspase-3 (dilution rate 1:1000) antibodies were purchased from Abcam (Cambridge, MA, United States), anti-B-cell lymphoma 2 (anti-Bcl-2, dilution rate 1:1000) and anti-Bcl-2-associated X protein (anti-Bax, dilution rate 1:1000) antibodies were purchased from Sangon Biotech (Shanghai, China), anti-cleaved caspase-9 antibody (dilution rate 1:1000) was purchased from CST Inc. (Danvers, MA, United States) and anti-GAPDH antibody (dilution rate 1:500) was purchased from Bioss Inc. (Woburn, MA, United States).

Techniques: Over Expression, In Vivo, Expressing, Western Blot, Staining, TUNEL Assay

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: De novo missense variants in RRAGC lead to a fatal mTORopathy of early childhood.

doi: 10.1016/j.gim.2023.100838

Figure Lengend Snippet: Figure 5 Subcellular localization of mTOR and biochemical analysis of published and novel variants in RRAGC. A. Patient (ID #1) and control (CT4) fibroblasts were stained with antibodies against mTOR and lysosomal-associated membrane protein 1 (Lamp1). Cells were cultured in the presence (+aa) or absence (−aa) of amino acids. Images show representative findings from 3 independent experiments. During the presence of amino acids, mTOR is mainly localized at the lysosomes. No clear differences between patient and control fibroblasts are visible under this condition. However, during amino acid starvation, mTOR redistributes to the cytosol in control fibroblasts. In contrast, mTOR remains mainly localized at the lysosomes in patient-derived fibroblasts. These findings suggest that the p.(Thr90Asn) variant de- couples mTOR activation from nutritional state. B. Representative western blot of protein lysates from HEK293 cells transfected with wild- type or mutant RRAGC incubated in media lacking L-leucine and L-methionine for 90 minutes. Whole-cell lysates were assessed for phosphorylated and total p70S6K and TFEB. B-actin was used as a loading control. The effect of the previously published variant p.(Ser75Tyr) was also assessed as a positive control. All novel variants were found to significantly increase S6 kinase and TFEB phos- phorylation compared with the wild type. C, D. Quantification of (C) phosphorylated relative to total p70S6K and (D) phosphorylated TFEB relative to total TFEB based on 3 independent biological replicates. Average values are presented as the mean ± SD. Significance was calculated using an ordinary one-way ANOVA in PRISM 9. *P < .05, **P < .01, ***P < .001, ****P < .0001. aa, amino acid; ANOVA, analysis of variance; mTOR, mechanistic target of rapamycin; TFEB, transcription factor EB.

Article Snippet: CoraLite 594- conjugated mTOR monoclonal antibody (mouse IgG2a; Proteintech CL594-66888; 1:100) and anti-LAMP1 monoclonal antibody (mouse IgG1, abcam ab25630; 1:100) were used as primary antibodies.

Techniques: Control, Staining, Membrane, Cell Culture, Derivative Assay, Variant Assay, Activation Assay, Western Blot, Transfection, Mutagenesis, Incubation, Positive Control

Journal: Nature Communications

Article Title: Structure of Myosin VI/Tom1 complex reveals a cargo recognition mode of Myosin VI for tethering

doi: 10.1038/s41467-019-11481-6

Figure Lengend Snippet: Biochemical analyses of the interaction between Myosin VI and Tom1. a A schematic diagram showing the domain arrangements of Myosin VI, Tom1, NDP52, TAX1BP1, and Optineurin. In this drawing, domains involved in the protein–protein interaction are highlighted with black lines, and the relevant interactions between two proteins are indicated by two-way arrows. b Superposition plots of the 1 H- 15 N HSQC spectra of Tom1(392–463) titrated with the un-labeled C-terminal CBD of Myosin VI proteins at different molar ratios. For clarity, the insert shows the enlarged view of a unique peak corresponding to the side chain of Tom1 W423 residue in the overlaid 1 H- 15 N HSQC spectra. c – e ITC-based measurements of the binding affinities of the C-terminal CBD of Myosin VI with Tom1(392–463) ( c ), Tom1(392–437) ( d ), and Tom1(437–463) ( e ). Kd values are the fitted dissociation constants with standard errors, when using the one-site binding model to fit the ITC data. ‘N.D.’ stands for that the Kd value is not detectable. Source data are provided as a Source Data file. f Overlay plots of the multi-angle light-scattering data of the C-terminal CBD of Myosin VI, Tom1(437–463), and the C-terminal CBD of Myosin VI in complex with Tom1(437–463). The derived molecular masses of the C-terminal CBD of Myosin VI and Tom1(437–463) are shown in red and in blue, respectively, while the derived molecular mass of the C-terminal CBD of Myosin VI and Tom1(437–463) complex is shown in black. The molecular masses errors are the fitted errors obtained from the data analysis software, and are showed in the brackets. The results clearly demonstrate that the C-terminal CBD of Myosin VI and Tom1(437–463) both form a stable monomer and may interact with each other to form a 1:1 stoichiometric complex in solution. Source data are provided as a Source Data file

Article Snippet: The prepared samples were separated by 4–15% gradient gels, blotted, and analyzed using antibodies to Myosin VI (proteintech, catalog no. 26778-1-AP, 1:1000 dilutions), Tom1 (Abcam, catalog no. ab99356, 1:1000 dilutions), TAX1BP1 (Abcam, catalog no. ab245636, 1:2000 dilutions), NDP52 (Abcam, catalog no. ab68588, 1:1000 dilutions) and Optineurin (Abcam, catalog no. ab213556, 1:1000 dilutions), respectively.

Techniques: Labeling, Residue, Binding Assay, Multi-Angle Light Scattering, Derivative Assay, Software

Journal: Nature Communications

Article Title: Structure of Myosin VI/Tom1 complex reveals a cargo recognition mode of Myosin VI for tethering

doi: 10.1038/s41467-019-11481-6

Figure Lengend Snippet: Statistics of ITC results between different variants of Myosin VI and Tom1

Article Snippet: The prepared samples were separated by 4–15% gradient gels, blotted, and analyzed using antibodies to Myosin VI (proteintech, catalog no. 26778-1-AP, 1:1000 dilutions), Tom1 (Abcam, catalog no. ab99356, 1:1000 dilutions), TAX1BP1 (Abcam, catalog no. ab245636, 1:2000 dilutions), NDP52 (Abcam, catalog no. ab68588, 1:1000 dilutions) and Optineurin (Abcam, catalog no. ab213556, 1:1000 dilutions), respectively.

Techniques:

Journal: Nature Communications

Article Title: Structure of Myosin VI/Tom1 complex reveals a cargo recognition mode of Myosin VI for tethering

doi: 10.1038/s41467-019-11481-6

Figure Lengend Snippet: Data collection and refinement statistics

Article Snippet: The prepared samples were separated by 4–15% gradient gels, blotted, and analyzed using antibodies to Myosin VI (proteintech, catalog no. 26778-1-AP, 1:1000 dilutions), Tom1 (Abcam, catalog no. ab99356, 1:1000 dilutions), TAX1BP1 (Abcam, catalog no. ab245636, 1:2000 dilutions), NDP52 (Abcam, catalog no. ab68588, 1:1000 dilutions) and Optineurin (Abcam, catalog no. ab213556, 1:1000 dilutions), respectively.

Techniques:

Journal: Nature Communications

Article Title: Structure of Myosin VI/Tom1 complex reveals a cargo recognition mode of Myosin VI for tethering

doi: 10.1038/s41467-019-11481-6

Figure Lengend Snippet: The overall structure of Myosin VI/Tom1 complex. a Ribbon diagram showing the overall structure of the C-terminal CBD of Myosin VI and Tom1 MBM complex. In this drawing, the C-terminal CBD of Myosin VI is shown in blue, and Tom1 MBM in magenta. b Surface representations showing the overall architecture of Myosin VI/Tom1 complex (left panel), and the open-book view of the binding interface between Myosin VI and Tom1 (right panel) with the same color scheme as in a

Article Snippet: The prepared samples were separated by 4–15% gradient gels, blotted, and analyzed using antibodies to Myosin VI (proteintech, catalog no. 26778-1-AP, 1:1000 dilutions), Tom1 (Abcam, catalog no. ab99356, 1:1000 dilutions), TAX1BP1 (Abcam, catalog no. ab245636, 1:2000 dilutions), NDP52 (Abcam, catalog no. ab68588, 1:1000 dilutions) and Optineurin (Abcam, catalog no. ab213556, 1:1000 dilutions), respectively.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Structure of Myosin VI/Tom1 complex reveals a cargo recognition mode of Myosin VI for tethering

doi: 10.1038/s41467-019-11481-6

Figure Lengend Snippet: The molecular interface of Myosin VI and Tom1 complex. a The combined surface representation and the ribbon-stick model showing the hydrophobic binding interface between the C-terminal CBD of Myosin VI and Tom1 MBM. In this presentation, the C-terminal CBD of Myosin VI is shown in the surface model and Tom1 MBM in the ribbon-stick model. Particularly, in the surface model of the C-terminal CBD of Myosin VI, the hydrophobic amino acid residues are drawn in yellow, the positively charged residues in blue, the negatively charged residues in red, and the uncharged polar residues in gray. b The combined surface charge representation and the ribbon-stick model showing the charge-charge interactions between the C-terminal CBD of Myosin VI and Tom1 MBM. c Stereo view of the ribbon-stick model showing the detailed interactions between the C-terminal CBD of Myosin VI and Tom1 MBM. The hydrogen bonds and salt bridges involved in the binding are shown as dotted lines. d Structure-based sequence alignment of Tom1 MBM with the corresponding regions of TomL1 and TomL2. In this structure-based sequence alignment, the conserved hydrophobic residues, polar neutral residues, positively charged residues, and negatively charged residues are colored in orange, green, blue, and magenta, respectively. Interface residues of Tom1 that are involved in the polar interactions and hydrophobic interactions with the C-terminal CBD of Myosin VI in the Myosin VI/Tom1 complex are further labeled with magenta stars and magenta triangles, respectively

Article Snippet: The prepared samples were separated by 4–15% gradient gels, blotted, and analyzed using antibodies to Myosin VI (proteintech, catalog no. 26778-1-AP, 1:1000 dilutions), Tom1 (Abcam, catalog no. ab99356, 1:1000 dilutions), TAX1BP1 (Abcam, catalog no. ab245636, 1:2000 dilutions), NDP52 (Abcam, catalog no. ab68588, 1:1000 dilutions) and Optineurin (Abcam, catalog no. ab213556, 1:1000 dilutions), respectively.

Techniques: Binding Assay, Sequencing, Labeling

Journal: Nature Communications

Article Title: Structure of Myosin VI/Tom1 complex reveals a cargo recognition mode of Myosin VI for tethering

doi: 10.1038/s41467-019-11481-6

Figure Lengend Snippet: Comparisons of the Myosin VI/Tom1 and Myosin VI/Dab2 complexes. a The comparison of the overall structures of the Myosin VI/Dab2 complex (gray-orange, PDB ID: 3H8D) and the Myosin VI/Tom1 complex (blue-magenta). In this presentation, the positions of site I and site II in the C-terminal CBD of Myosin VI are further indicted. b Stereo view in the ribbon-stick model showing the comparison of the binding interfaces of the Myosin VI/Dab2 complex and the Myosin VI/Tom1 complex with the same color scheme as in a . The hydrogen bonds and salt bridges involved in the interactions are shown as dotted lines. The binding interface residues of Tom1 and Dab2 are labeled with magenta and orange numbers, respectively. While, the interface residues of the C-terminal CBD of Myosin VI that involved in the interactions with both Tom1 and Dab2, only for the interaction with Tom1 or Dab2, are labeled with black, blue, and gray numbers, respectively. c Structure-based sequence alignment of Tom1(437–493) and the Myosin VI-binding region of Dab2. In this structure-based sequence alignment, the conserved hydrophobic residues, polar neutral residues, positively charged residues and negatively charged residues are colored in orange, green, blue, and magenta, respectively. Key interface residues of Tom1 involved in the interaction with the site I of the C-terminal CBD of Myosin VI through the polar interactions and the hydrophobic interactions are further labeled with magenta stars and triangles, respectively, and that of Dab2 are highlighted with orange stars and triangles. Meanwhile, key interface residues of Dab2 that are critical for binding to the site II of the C-terminal CBD of Myosin VI, are labeled with orange dots

Article Snippet: The prepared samples were separated by 4–15% gradient gels, blotted, and analyzed using antibodies to Myosin VI (proteintech, catalog no. 26778-1-AP, 1:1000 dilutions), Tom1 (Abcam, catalog no. ab99356, 1:1000 dilutions), TAX1BP1 (Abcam, catalog no. ab245636, 1:2000 dilutions), NDP52 (Abcam, catalog no. ab68588, 1:1000 dilutions) and Optineurin (Abcam, catalog no. ab213556, 1:1000 dilutions), respectively.

Techniques: Comparison, Binding Assay, Labeling, Sequencing

Journal: Nature Communications

Article Title: Structure of Myosin VI/Tom1 complex reveals a cargo recognition mode of Myosin VI for tethering

doi: 10.1038/s41467-019-11481-6

Figure Lengend Snippet: Myosin VI can link Tom1 with autophagy receptors. a A co-immunoprecipitation assay showing that point mutations of key interface residues observed in the Myosin VI/Tom1 complex structure abolish the specific interaction between Myosin VI(1060–1285) and Tom1 in cells. In this assay, cell extracts were prepared from HEK293T cells co-transfected with different combinations of plasmids as indicated, and 5% of each extracts were used as loading controls (bottom panel). b A co-immunoprecipitation assay revealing that Myosin VI(1060–1285), Tom1 and autophagy receptor TAX1BP1, NDP52, or Optineurin, can form ternary complexes in co-transfected cells. c A co-immunoprecipitation assay showing that mutations of Tom1, which can disrupt the interaction between Myosin VI(1060–1285) and Tom1, can also abolish the formation of the Tom1/Myosin VI/autophagy receptor ternary complex in cells. In this assay, cell extracts were prepared from HEK293T cells co-transfected with different combinations of plasmids as indicated, and 5% of each extracts were used as loading controls (bottom panel). Source data are provided as a Source Data file. d A proposed model depicting the tethering of endosome and autophagosome mediated by Myosin VI in cooperate with Tom1, the autophagy receptors, NDP52, TAX1BP1, and Optineurin as well as relevant ubiquitin chains, for facilitating the maturation of autophagosome in autophagy

Article Snippet: The prepared samples were separated by 4–15% gradient gels, blotted, and analyzed using antibodies to Myosin VI (proteintech, catalog no. 26778-1-AP, 1:1000 dilutions), Tom1 (Abcam, catalog no. ab99356, 1:1000 dilutions), TAX1BP1 (Abcam, catalog no. ab245636, 1:2000 dilutions), NDP52 (Abcam, catalog no. ab68588, 1:1000 dilutions) and Optineurin (Abcam, catalog no. ab213556, 1:1000 dilutions), respectively.

Techniques: Co-Immunoprecipitation Assay, Transfection, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Mechanisms underlying melanoma invasion as a consequence of MLK3 loss

doi: 10.1101/2021.12.10.472116

Figure Lengend Snippet: a) LOX cells stably expressing shMLK3 were transfected with mCherry-MT1-MMP (red) plasmid assessed for invasion on FITC-labeled gelatin (green). Images along the x/y and x/z axes are shown. b-c) LOX cells were infected with lentivirus carrying GFP-shMLK3 plasmid, plated on unlabeled gelatin and either treated with PD98059 inhibitor or mock treated. Cells labeled for endogenous MT1-MMP and actin. MT1-MMP pixel intensity was measured using ImageJ. The amount of MT1-MMP in contact with gelatin was determined by the percentage of basal fluorescence of MT1-MMP divided by the total cell fluorescence. d-e) Lysates from shMLK3-infected cells or shMLK3-infected cells treated with PD98059 inhibitor were immunoprecipitated using anti-phosphoserine or TOM1L1 antibodies. Whole cell lysates and immunoprecipitates were resolved by SDS-PAGE and probed with the appropriate antibodies as indicated by Western blotting (d). Proteins bans were quantitated using ImageJ (e). The error bars indicate standard error. **p-value<0.01, ****p-value<0.001

Article Snippet: The following primary antibodies were used in this study: MLK3 antibody (Abcam), phospho-ERK, ERK, phopsho-JNK, JNK, beta-actin (Cell signaling Technologies), Raf-B and Cdc37 antibodies (Santa Cruz Technologies), phosphoserine (Millipore), alpha-tubulin (Sigma Aldrich) and TOM1L1 (Proteintech).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Labeling, Infection, Fluorescence, Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Mechanisms underlying melanoma invasion as a consequence of MLK3 loss

doi: 10.1101/2021.12.10.472116

Figure Lengend Snippet: The data described in this study shows that inhibition of MLK3 results in increased melanoma invasion. Loss of MLK3 expression results in the hyperactivation of ERK, which is linked to the formation of a BRAF/Hsp90/Cdc37 protein complex. Enhanced ERK activation, leads to subsequent inactivation of GSK3β, and increased stability and activation of JNK. ERK activation also prompts transcription of MT1-MMP and the serine-phosphorylation of TOM1L1 leading to localization of MT1-MMP to invadopodia structures.

Article Snippet: The following primary antibodies were used in this study: MLK3 antibody (Abcam), phospho-ERK, ERK, phopsho-JNK, JNK, beta-actin (Cell signaling Technologies), Raf-B and Cdc37 antibodies (Santa Cruz Technologies), phosphoserine (Millipore), alpha-tubulin (Sigma Aldrich) and TOM1L1 (Proteintech).

Techniques: Inhibition, Expressing, Activation Assay, Phospho-proteomics

Journal: Aging (Albany NY)

Article Title: Geniposide-mediated protection against amyloid deposition and behavioral impairment correlates with downregulation of mTOR signaling and enhanced autophagy in a mouse model of Alzheimer's disease

doi: 10.18632/aging.101759

Figure Lengend Snippet: Geniposide treatment decreases mTOR activation markers in brains of APP/PS1 mice. Hippocampal expression of Akt, mTOR, and 4E-BP1, and their respective phosphorylated forms was detected by western blot. The expression of p-Akt ( A ) and p-mTOR ( B ) was enhanced in APP/PS1 mice compared to WT, and geniposide attenuated this increase. The expression of p-4E-BP1 ( C ) in APP/PS1 mice was reduced compared to WT, and geniposide partly restored this decrease. Data are presented as mean ± SEM (n = 6). *** p < 0.001, ** p < 0.01, * p < 0.05 vs. WT; # p < 0.05 vs. APP/PS1 mice (one-way ANOVA, Tukey's Multiple Comparison Test). WT: wild-type mice. GP: geniposide.

Article Snippet: The membranes were blocked in 5% bovine serum albumin in TBST (Tris-buffered saline with 0.05% Tween-20) for 1h, and incubated overnight at 4°C with primary antibodies directed against: Akt (1:1,000), p-Akt (1:2,000), mTOR (1:1,000), p-mTOR (1:1,000), 4E-BP1 (1:1,000), or p-4E-BP1 (1:2,000), followed by incubation at 4°C for 2h with a goat-anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China).

Techniques: Activation Assay, Expressing, Western Blot, Comparison