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: bioRxiv

Article Title: Chemical inhibition of pathological reactive astrocytes promotes neural protection

doi: 10.1101/2021.11.03.467083

Figure Lengend Snippet: a , Expression of cell-type specific markers layered onto UMAP plots from single-cell RNAseq of primary resting astrocytes. b , UMAP plot of resting and pathological reactive astrocytes (abbreviated to reactive in figures) single-cell RNAseq colored by condition, resting (grey) and reactive (red). Additional UMAP plots showing expression levels for the pathological reactive astrocyte markers C3 and Gbp2 . c , Representative images of in situ hybridization in resting and reactive astrocyte cultures with probes against the pathological reactive astrocyte markers C3 and Serping1 . d-f , Gene set enrichment analysis (GSEA) comparing pathological reactive astrocytes to the top 100 genes upregulated in astrocytes from single-nuclei RNAseq data from d , Alzheimer’s, e , Huntington’s, and f , Parkinson’s disease patient tissue. g , Representative images of resting and reactive astrocyte cultures exposed to the OVA 257-264 peptide and then stained for MHC Class I bound to OVA 257-264 (H-2Kb+OVA 257-264 ) in red. h , The Log2 fold-change (Log2FC) of secreted cytokines in reactive vs resting astrocytes conditioned media. Data presented as mean ± s.e.m for n = 2 biological replicates (independent astrocyte isolations). i , Scatter plot of primary screen results displayed as percent GBP2 positive, normalized to reactive astrocyte plus vehicle controls for all non-toxic chemicals and validated hit chemicals colored in blue. The dashed blue line represents the hit cut-off at a ≥90% decrease in GBP2-positive astrocytes compared to reactive astrocyte plus vehicle controls. Dashed red line represents the average percent GBP2 positive for reactive astrocytes plus vehicle set at 100%. Solid lines represent +/- 2 standard deviations from the mean of reactive plus vehicle control wells. j , Pie chart depicting the chemical class breakdown of all 29 validated chemical hits.

Article Snippet: After incubation for 24 hours, the cells were fixed using 4% paraformaldehyde and stained for GBP2 (Proteintech #11854-1-AP) using the procedure detailed in the immunocytochemistry section below, then imaged using the PerkinElmer Operetta CLS High-Content Analysis System.

Techniques: Expressing, In Situ Hybridization, Staining, Control

Journal: bioRxiv

Article Title: Chemical inhibition of pathological reactive astrocytes promotes neural protection

doi: 10.1101/2021.11.03.467083

Figure Lengend Snippet: a , Example images of DMSO vehicle-treated reactive and resting control wells. Scale bar is 100um. b , Z-prime standard, and robust scores for each primary screen plate. c , Average percent GBP2 positive astrocytes in DMSO vehicle treated reactive and resting control wells on each primary screen plate. d , Dose curve analysis of hits from primary screen. Compounds were tested across an 8-point dose curve with decreasing half-steps from 6uM to 0.05uM. Data are presented as the percent of GBP2 positive cells normalized to DMSO vehicle treated reactive astrocyte control wells. n = 2 biological replicates (independent astrocyte isolations). Black data points represent toxic doses where total cell number in the well decreased by >50% compared to DMSO vehicle treated reactive astrocyte control wells. e , Dose curve analysis of hits from primary screen with Psmb8 positivity by in situ hybridization as a secondary endpoint. Compounds were tested across an 8-point dose curve with decreasing half-steps from 6uM to 0.05uM. Data are presented as the percent of Psmb8 positive normalized to DMSO vehicle treated reactive astrocyte control wells with an n = 1 biological replicate (independent astrocyte isolation). Black data points represent toxic doses where total cell number in the well decreased by >50% compared to DMSO vehicle treated reactive astrocyte control wells.

Article Snippet: After incubation for 24 hours, the cells were fixed using 4% paraformaldehyde and stained for GBP2 (Proteintech #11854-1-AP) using the procedure detailed in the immunocytochemistry section below, then imaged using the PerkinElmer Operetta CLS High-Content Analysis System.

Techniques: Control, In Situ Hybridization, Isolation

Journal: bioRxiv

Article Title: Chemical inhibition of pathological reactive astrocytes promotes neural protection

doi: 10.1101/2021.11.03.467083

Figure Lengend Snippet: a , Ranked inhibition against each HDAC isozyme for the validated HDAC inhibitors identified in the primary screen. Highlighted is HDAC3, which is the only shared target between all HDAC inhibitor hits from the primary screen. Ranked efficiency was pulled from target data provided by Selleck Chemical for each compound. b , Dose curve and IC50 value for the HDAC3 specific inhibitor RGFP966. c , Dose curve and IC50 value for the HDAC3 specific inhibitor T247. d , Representative images of wild-type (WT) and HDAC3 knockout (KO) astrocyte cultures exposed to the reactive factors TNF, IL1a, and C1q. Scale bar is 100um. e , Quantification of the percentage of cells positive for HDAC3 normalized to WT for the experiment represented in d. Data are presented as the mean ± s.e.m. for n = 3 biological replicates (independent astrocyte isolations). p-value generated with a Student’s two-tailed t-test. f , Quantification of the percentage of cells positive for GBP2 normalized to WT for the experiment represented in d. Data are presented as the mean ± s.e.m. for n = 3 biological replicates (independent astrocyte isolations). p-value generated with a Student’s two-tailed t-test. g , GBP2 and PSMB8 qPCR results for human iPSC derived resting or reactive (TNF, IL1α, and C1q treated) astrocyte cultures treated with vehicle or 5uM RGFP966. Data are presented as mean ± s.e.m. for n = 4 technical replicates.

Article Snippet: After incubation for 24 hours, the cells were fixed using 4% paraformaldehyde and stained for GBP2 (Proteintech #11854-1-AP) using the procedure detailed in the immunocytochemistry section below, then imaged using the PerkinElmer Operetta CLS High-Content Analysis System.

Techniques: Inhibition, Knock-Out, Generated, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: Chemical inhibition of pathological reactive astrocytes promotes neural protection

doi: 10.1101/2021.11.03.467083

Figure Lengend Snippet: a) Representative images of Gbp2 (red) and Gfap (blue) in situ hybridization from the frontal cortex and corpus callosum of mice after 48hrs of systemic lipopolysaccharide (LPS) exposure and treated with vehicle or 10mg/kg RGFP. Scale bar is 100um. b-c , Quantification of in situ data represented in a. The percentage of astrocytes that are GBP2+ in the b , frontal cortex or c , corpus callosum of mice exposed to LPS and treated with vehicle or 10mg/kg RGFP is presented as mean ± s.e.m for n = 4 biological replicates (2 male and 2 female mice) with p-value generated by Student’s unpaired two-tailed t-test. d , Representative images of C3 and Slc1a3 in situ hybridization in the dorsal column of lysolecithin (LPC) lesioned mice at 12 days post lesion and treated with vehicle or 10mg/kg RGFP. Scale bar is 50um. e , Quantification of in situ data represented in D. The percent of astrocytes that are C3 positive in LPC-exposed mice treated chronically with vehicle 10mg/kg RGFP. Data are presented as the mean ± s.e.m for n = 4-6 biological replicates (mice) with p-value generated by Student’s unpaired two-tailed t-test. f , Representative electron microscopy (EM) images of LPC lesioned mice treated with vehicle or 10mg/kg RGFP. Scale bar is 10um. g , Quantification of axon density in EM images represented in f. Total axon density normalized to control for LPC lesioned mice treated with vehicle ( n = 4 mice) or 10mg/kg RGFP ( n = 3 mice) is presented as mean ± s.e.m. with p-value generated by Student’s unpaired two-tailed t-test. h) Quantification of remyelinated axon density in EM images represented in f. Myelinated axon density normalized to control for LPC lesioned mice treated with vehicle or 10mg/kg RGFP is presented as mean ± s.e.m for n = 3-4 biological replicates (mice) with p-value generated by Student’s unpaired two-tailed t-test.

Article Snippet: After incubation for 24 hours, the cells were fixed using 4% paraformaldehyde and stained for GBP2 (Proteintech #11854-1-AP) using the procedure detailed in the immunocytochemistry section below, then imaged using the PerkinElmer Operetta CLS High-Content Analysis System.

Techniques: In Situ Hybridization, In Situ, Generated, Two Tailed Test, Electron Microscopy, Control