Journal: STAR Protocols

Article Title: Measuring microtubule binding kinetics of membrane-bound kinesin motors using supported lipid bilayers

doi: 10.1016/j.xpro.2021.100691

Figure Lengend Snippet:

Article Snippet: Guanosine 5′-triphosphate, disodium salt trihydrate (GTP) , Jena Bioscience , Cat#: NU-1012-10G.

Techniques: Recombinant, Electron Microscopy, Software, Fluorescence, Microscopy

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting AGTPBP1 inhibits pancreatic cancer progression via regulating microtubules and ERK signaling pathway.

doi: 10.1186/s10020-024-00892-x

Figure Lengend Snippet: Fig. 1 Expression of AGTPBP1 in PDAC tissues. A HE staining of cancer tissue and precancerous normal tissues of PDAC patients was conducted. Scale bar, 100 μm. B Immunohistochemical staining using anti-AGTPBP1 antibody showed higher expression in PDAC than normal pancreatic tissues. Scale bar, 100 μm. C The average optical density (AOD) of AGTPBP1 immunohistochemical staining in the cancer tissues of 40 PDAC patients was measured. D AGTPBP1 immunohistochemical staining showed that it was mainly expressed in pancreatic islets in normal pancreatic tissues, while it was mainly expressed in pancreatic ductal epithelial cells in PDAC tissues. Scale bar, 100 μm. E Expression of AGTPBP1 in 40 PDAC and 19 normal pancreatic tissues. F Expression of AGTPBP1 in 19 pairs of PDAC and normal pancreatic tissues. All data are presented as means ± SD. The Student’s t-test was used to compare the means between the two groups. ***P < 0.001 versus (vs.) normal group

Article Snippet: After that, they were incubated at 4 °C overnight with the specified primary antibodies as follows: AGTPBP1 (1:1000), GAPDH (1:5000), ERK1/2(1:1000), phospho-ERK1/2 (P-ERK1/2, 1:1000) and TUBB4B (1:1000) from ProteinTech, USA.

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting AGTPBP1 inhibits pancreatic cancer progression via regulating microtubules and ERK signaling pathway.

doi: 10.1186/s10020-024-00892-x

Figure Lengend Snippet: Fig. 2 Expression of AGTPBP1 in human pancreatic cancer cell lines and siRNA inhibition of AGTPBP1 expression in PANC-1 cells. A The expres sion of AGTPBP1 was up-regulated in PANC-1 and HPAF-II PC cells compared with normal control pancreatic endothelial cells by RT-qPCR. **P < 0.01, ****P < 0.0001 vs. HPDE6-C7. B-C The siRNA-1(targeting AGTPBP1-1050) and siRNA-2 (targeting AGTPBP1-2136) could inhibit AGTPBP1 expression. B The relative expression of AGTPBP1 by RT-qPCR. C Western blot analysis of AGTPBP1 expression. All data are presented as means ± SD. The Student’s t-test was used for statistical comparison. All experiments were performed in triplicate. **P < 0.0001, ****P < 0.01 vs. sh-NC

Article Snippet: After that, they were incubated at 4 °C overnight with the specified primary antibodies as follows: AGTPBP1 (1:1000), GAPDH (1:5000), ERK1/2(1:1000), phospho-ERK1/2 (P-ERK1/2, 1:1000) and TUBB4B (1:1000) from ProteinTech, USA.

Techniques: Expressing, Inhibition, Control, Quantitative RT-PCR, Western Blot, Comparison

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting AGTPBP1 inhibits pancreatic cancer progression via regulating microtubules and ERK signaling pathway.

doi: 10.1186/s10020-024-00892-x

Figure Lengend Snippet: Fig. 3 Knockdown of AGTPBP1 inhibits the malignant biological behaviors of pancreatic cancer cells in vitro A The proliferation experiments using CCK-8 cells showed that knocking out AGTPBP1 in PANC-1 cells could inhibit its proliferation ability (n = 5). B Plate clonalization experiments showed that knocking out AGTPBP1 significantly inhibited the formation of colonies of pancreatic cancer cells, counting the number of colonies containing more than 50 cells (n = 3). Scale bar, 200 μm. C Cell scratch healing experiments showed that knocking out AGTPBP1 significantly inhibited the migration ability of pancreatic cancer cells (n = 3). Scale bar, 200 μm. Transwell compartment migration and invasion experiments showed that knocking down AGTPBP1 could significantly reduce the migration (D) and invasion (E) ability of pancreatic cancer cells (n = 3). Scale bar, 200 μm. F After knocking out AGTPBP1 for pancreatic cancer cells, cells are blocked in the G2/M phase (n = 3). All results are representative of three independent experiments. Values are presented as mean ± SD. The Student’s t-test was used to compare the means between the two groups. **P<0.01,***P<0.001,****P<0.0001 vs. sh-NC

Article Snippet: After that, they were incubated at 4 °C overnight with the specified primary antibodies as follows: AGTPBP1 (1:1000), GAPDH (1:5000), ERK1/2(1:1000), phospho-ERK1/2 (P-ERK1/2, 1:1000) and TUBB4B (1:1000) from ProteinTech, USA.

Techniques: Knockdown, In Vitro, CCK-8 Assay, Migration

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting AGTPBP1 inhibits pancreatic cancer progression via regulating microtubules and ERK signaling pathway.

doi: 10.1186/s10020-024-00892-x

Figure Lengend Snippet: Fig. 4 Knockdown of AGTPBP1 can inhibit the growth of xenograft tumors and the formation of new blood vessels in pancreatic cancer cells. A Comparative tumor manifestations and volume of subcutaneous tumorigenesis in the PANC-1 cell line AGTPBP1 KD group (n = 5) and the control group (n = 6). B-D Representative images from IHC staining of AGTPBP1 (B), Ki67 (C), and CD31 (D) expression in xenograft tumors. The positive signal was measured by ImageJ. Scale bar, 100 μm. Values are presented as mean ± SD. The Student’s t-test was used to compare the means between the two groups. *(P < 0.05), **P<0.01 vs. sh-NC

Article Snippet: After that, they were incubated at 4 °C overnight with the specified primary antibodies as follows: AGTPBP1 (1:1000), GAPDH (1:5000), ERK1/2(1:1000), phospho-ERK1/2 (P-ERK1/2, 1:1000) and TUBB4B (1:1000) from ProteinTech, USA.

Techniques: Knockdown, Control, Immunohistochemistry, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting AGTPBP1 inhibits pancreatic cancer progression via regulating microtubules and ERK signaling pathway.

doi: 10.1186/s10020-024-00892-x

Figure Lengend Snippet: Fig. 5 The differentially expressed genes in the AGTPBP1 KD cells compared to control cells through RNA-seq and bioinformatic analysis.A A volcano map of differentially expressed genes in the AGTPBP1 knockdown and control groups (n = 3/group). Red dots indicate upregulated genes and blue dots indicate downregulated genes. B The heat map with significant expression differences between the AGTPBP1 knockdown group and the control group (n = 3/group). Red: up-regulated DEGs; blue: down-regulated DEGs. The redder the color, the higher the expression, and the greener, the lower the expression. C GO enrichment analysis of significantly differentially expressed genes in the AGTPBP1 knockdown group and the control group cells (n = 3/group). D Bubble plot of KEGG enrichment analysis of significantly differentially expressed genes in the AGTPBP1 knockdown and control groups (n = 3/group). The size of the dot represents the number of genes annotated, and the color from green to red represents the degree of enrichment. P?0.05 is considered significantly different.

Article Snippet: After that, they were incubated at 4 °C overnight with the specified primary antibodies as follows: AGTPBP1 (1:1000), GAPDH (1:5000), ERK1/2(1:1000), phospho-ERK1/2 (P-ERK1/2, 1:1000) and TUBB4B (1:1000) from ProteinTech, USA.

Techniques: Control, RNA Sequencing, Knockdown, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting AGTPBP1 inhibits pancreatic cancer progression via regulating microtubules and ERK signaling pathway.

doi: 10.1186/s10020-024-00892-x

Figure Lengend Snippet: Fig. 6 Expression of MYLK and MAP1A in PC and AGTPBP1 KD cells.A-B MYLK (A) and MAP1A (B) were up-regulated in pancreatic cancers based on GEPIA analysis. PAAD, pancreatic ductal adenocarcinoma; T, tumor; N, normal. C-D Both MYLK (C) and MAP1A (D) were down-regulated after AGTPBP1 knockdown in PANC-1 PC cells by RT-qPCR analysis. n = 3. E-F Positive correlations between the expression of AGTPBP1 and MYLK, AGTPBP1 and MAP1A in PDAC samples were observed by GEPIA analysis. Spearman correlation was used to measure the degree of association between two genes. Values are presented as mean ± SD. The Student’s t-test was used to compare the means between the two groups. *P<0.05, **P<0.01, ****P<0.0001 vs. sh-NC

Article Snippet: After that, they were incubated at 4 °C overnight with the specified primary antibodies as follows: AGTPBP1 (1:1000), GAPDH (1:5000), ERK1/2(1:1000), phospho-ERK1/2 (P-ERK1/2, 1:1000) and TUBB4B (1:1000) from ProteinTech, USA.

Techniques: Expressing, Knockdown, Quantitative RT-PCR

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting AGTPBP1 inhibits pancreatic cancer progression via regulating microtubules and ERK signaling pathway.

doi: 10.1186/s10020-024-00892-x

Figure Lengend Snippet: Fig. 7 The expression of TUBB4B, ERK1/2, and p-ERK1/2 was down-regulated in PANC-1 cells after AGTPBP1 knockdown. A mRNA level of TUBB4B in AGTPBP1 KD PANC-1 and control cells was detected by RT-qPCR. GAPDH was an internal control. n = 3. B Protein level of AGTPBP1, TUBB4B, ERK1/2, and p-ERK1/2 in AGTPBP1 KD PANC-1 and control cells was detected by western blot. GAPDH was an internal control. n = 3. C The gray density of the immunoblot was estimated and normalized with GAPDH to demonstrate the relative expression level of proteins in B. As a result, the knockdown of AGTPBP1 in PANC-1 cells significantly inhibited the expression of TUBB4B, ERK1/2, and p-ERK1/2. Values are presented as mean ± SD. The Student’s t-test was used to compare the means between the two groups. *P<0.05 , **P<0.01 ,*** P<0.001, **** P<0.0001 vs. sh-NC

Article Snippet: After that, they were incubated at 4 °C overnight with the specified primary antibodies as follows: AGTPBP1 (1:1000), GAPDH (1:5000), ERK1/2(1:1000), phospho-ERK1/2 (P-ERK1/2, 1:1000) and TUBB4B (1:1000) from ProteinTech, USA.

Techniques: Expressing, Knockdown, Control, Quantitative RT-PCR, Western Blot

Journal: Molecular Pharmacology

Article Title: Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2?,3?- O -( N -Methylanthraniloyl)-Inosine 5?-Triphosphate S⃞

doi: 10.1124/mol.111.071894

Figure Lengend Snippet: Structure of MANT nucleoside 5′-triphosphates (NTPs). Represented are MANT-ITP, MANT-GTP, and MANT-XTP, the MANT nucleotides used for enzymatic studies, fluorescence spectroscopy, crystallography, and structure activity evaluation. The MANT group isomerizes between the 2′ and 3′-O-ribosyl function. Note the different substitution of the C2 carbon atom of the purine ring in the various nucleotides.

Article Snippet: MANT-GTP and MANT-XTP were obtained from Jena Bioscience (Jena, Germany).

Techniques: Fluorescence, Spectroscopy, Activity Assay

Journal: Molecular Pharmacology

Article Title: Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2?,3?- O -( N -Methylanthraniloyl)-Inosine 5?-Triphosphate S⃞

doi: 10.1124/mol.111.071894

Figure Lengend Snippet: Fluorescence emission spectra of MANT-GTP, MANT-ITP and MANT-XTP. Emission at λex = 280 nm (λem = 300–500 nm) and at λex = 350 nm (λem = 370–500 nm) are represented. Experiments were conducted at 25°C. Addition of MANT nucleotides (1 μM), blue lines; subsequent addition of VC1 (5 μM) and IIC2 (25 μM), green lines; subsequent addition of FS (100 μM), red lines. Dashed lines in A to C represent endogenous tyrosine/tryptophan fluorescence of VC1:IIC2. Reaction mixtures contained a buffer of 100 mM KCl, 10 mM MnCl2, and 25 mM HEPES/NaOH, pH 7.4. Three independent experiments with at least two different batches of VC1:IIC2 were performed. A and B, MANT-GTP; C and D, MANT-ITP; E and F, MANT-XTP. Fluorescence intensities are shown in arbitrary units (a.u.). For FRET (A, C, and E, λex = 280 nm), the fluorescence observed with VC1:IIC2 was set to 100%. In direct fluorescence experiments (B, D, and F, λex = 350 nm), the fluorescence observed with MANT nucleotides alone was set to 100%.

Article Snippet: MANT-GTP and MANT-XTP were obtained from Jena Bioscience (Jena, Germany).

Techniques: Fluorescence

Journal: Molecular Pharmacology

Article Title: Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2?,3?- O -( N -Methylanthraniloyl)-Inosine 5?-Triphosphate S⃞

doi: 10.1124/mol.111.071894

Figure Lengend Snippet: Inhibitory potencies of MANT-NTPs on the catalytic activity of VC1:IIC2 Catalytic activities of VC1:IIC2 were determined as described under Materials and Methods . Reactions were conducted in the presence of 10 mM MnCl 2 and 100 μM FS in the absence or presence of G s α-GTPγS. Data were analyzed by nonlinear regression to calculate K i values. The catalytic activity of C1/C2 in the presence of Mn 2+ + FS + G s α-GTPγS with 100 μM ATP as substrate was 2700 ± 350 nmol · mg −1 · min −1 and in the presence of Mn 2+ + FS, the activity was 300 ± 110 nmol · mg −1 · min −1 . The K m values for VC1:IIC2 were reported previously ( Mou et al., 2005 ) for each experimental condition (430 and 620 μM, respectively) and were used to calculate K i values from IC 50 values. Data are the mean values ± S.D. of two to four independent experiments performed in duplicates with at least two different batches of protein.

Article Snippet: MANT-GTP and MANT-XTP were obtained from Jena Bioscience (Jena, Germany).

Techniques: Activity Assay

Journal: Molecular Pharmacology

Article Title: Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2?,3?- O -( N -Methylanthraniloyl)-Inosine 5?-Triphosphate S⃞

doi: 10.1124/mol.111.071894

Figure Lengend Snippet: Binding mode of MANT-ITP and two Mn2+ ions in the catalytic site. MANT-ITP and two metal ions are bound in the cleft between the soluble C1a and C2a domains. VC1 and IIC2 are colored wheat and light pink, respectively. MANT-ITP is shown as stick model, carbon atoms are cyan, nitrogen atoms are dark blue, oxygen atoms are red, and phosphorus atoms are green. The two Mn2+ ions are shown as orange spheres. A, difference electron density for 3′-O-MANT-ITP and Mn2+. The lime green wire represents the |Fo|-|Fc| electron density for MANT-ITP contoured at 2.5 σ. The blue wire corresponds to the |Fo|-|Fc| electron density for the two Mn2+ ions contoured at 5 σ. The coordinates for the ligands were omitted from the phasing model. The secondary structure elements of the complex are labeled as defined previously (Tesmer et al., 1997). B, detailed view of substrate binding site of VC1:IIC2 with MANT-ITP:Mn2+. The catalytic site of VC1:IIC2 shows MANT-ITP, A- and B- site of two Mn2+ ions and the protein residues that are responsible for ligand interaction. The interaction among protein residues and MANT-ITP, Mn2+ are shown as dashed gray lines. C, superimposed crystal structures of 3′-O-MANT-ITP and 3′-O-MANT-GTP. The derived MANT-ITP crystal structure was superimposed and compared with the crystal structure of MANT-GTP, shown as a transparent yellow stick model (Protein Data Bank code 1TL7) (Mou et al., 2005). The protein residues are in almost identical conformation, and the inhibitors are situated in the substrate binding pocket in a similar fashion. D, superimposed purine binding site of 3′-O-MANT-ITP and 3′-O-MANT-GTP. The interaction of the hypoxanthine ring and guanine ring of MANT-ITP and MANT-GTP are shown as dashed black and olive green lines, respectively. The distances of hydrogen bond between the hypoxanthine ring and surrounding protein residues of MANT-ITP are indicated in Ångstroms. The hydrogen bond between Ile1019 and the amino group of MANT-GTP is missing in the MANT-ITP structure. Lys938 and the oxygen of the hypoxanthine ring are further apart. The hypoxanthine ring has less binding constraint in the purine binding pocket in comparison to the guanine ring of MANT-GTP.

Article Snippet: MANT-GTP and MANT-XTP were obtained from Jena Bioscience (Jena, Germany).

Techniques: Binding Assay, Labeling, Derivative Assay

Journal: Molecular Pharmacology

Article Title: Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2?,3?- O -( N -Methylanthraniloyl)-Inosine 5?-Triphosphate S⃞

doi: 10.1124/mol.111.071894

Figure Lengend Snippet: MANT-binding site. A detailed view of the MANT-binding site is depicted. MANT-ITP is shown as a stick model; carbon atoms are cyan, nitrogen atoms are dark blue, oxygen atoms are red and one phosphorus atom is displayed in green. VC1 and IIC2 are colored wheat and light pink, respectively. MANT-GTP is shown as a transparent yellow stick model. The carbonyl group of MANT-ITP is in closer contact to Asn1025 but does not interact with the side chain of Asn1025 in this orientation. Apart from this, no conformational differences between MANT-ITP and MANT-GTP are detected. However, MANT-ITP might exert stronger hydrophobic interactions due to changes of the relative positions of Trp1020 and the MANT group.

Article Snippet: MANT-GTP and MANT-XTP were obtained from Jena Bioscience (Jena, Germany).

Techniques: Binding Assay

Journal: Molecular Pharmacology

Article Title: Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2?,3?- O -( N -Methylanthraniloyl)-Inosine 5?-Triphosphate S⃞

doi: 10.1124/mol.111.071894

Figure Lengend Snippet: Comparison of the binding of MANT-ITP and MANT-GTP by molecular dynamics simulations. Overlaid graphical representations of the terminal (t = 9.6 ns) time steps for the MANT-GTP (CPK-colored sticks with green carbons and orange phosphorus atoms) and MANT-ITP (CPK-colored sticks with cyan carbons and tan phosphorus atoms) interacting with the VC1:IIC2 receptor (pale green ribbons) and its cofactor Mn2+ ions (magenta spheres for MANT-GTP simulation and purple for MANT-ITP). Additional details on differences in the interactions of MANT-GTP and MANT-ITP with VC1:IIC2 are provided in Supplemental Fig. 1 and Supplemental Tables 1 to 3.

Article Snippet: MANT-GTP and MANT-XTP were obtained from Jena Bioscience (Jena, Germany).

Techniques: Binding Assay

Journal: Current biology : CB

Article Title: Collective Force Regulation in Anti-Parallel Microtubule Gliding by Dimeric Kif15 Kinesin Motors

doi: 10.1016/j.cub.2017.08.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GTP , Cytoskeleton , Cat#BST06.

Techniques: Virus, Expressing, Recombinant, Software