Journal: International Journal of Biological Sciences

Article Title: Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy

doi: 10.7150/ijbs.62393

Figure Lengend Snippet: MTX1 was identified as a sorafenib resistance factor by Genome-Scale CRISPRa Screening. (A) Flow chart of CRISPR-Pool™SAM human library screening for genes involving in sorafenib resistance in HCC-LM3 cells. (B) Scatterplot displayed enrichment of sgRNAs in surviving cells after sorafenib treatment compared to the control group. (C) Expression boxplot of LENEP, BCL11B, BAG3, RANBP1, and MTX1 in human HCC patients from GEPIA online database ( http://gepia.cancer-pku.cn/ ). Red boxes indicate tumor tissue and gray boxes represent normal tissue. (D) Kaplan-Meier survival curves were analyzed for above candidate genes based on UALCAN ( http://ualcan.path.uab.edu/analysis.html ). (E) The mRNA expression level of MTX1 in 40 pairs of HCC and adjacent normal samples were determined by qRT-PCR. The data of qRT-PCR are presented as mean ± SD from three experiments, **P < 0.01.

Article Snippet: MTX1 antibody (15529-1-AP, Proteintech, Wuhan, China) was used to examine MTX1 expression in the HCC tissue microarray.

Techniques: CRISPR, Library Screening, Control, Expressing, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy

doi: 10.7150/ijbs.62393

Figure Lengend Snippet: Enforced expression of MTX1 accelerated HCC cell growth. (A) Overexpression of MTX1 was confirmed by western blot. (B) CCK-8 assays were performed using cells with or without stable MTX1 overexpression, and cell growth curves were shown. (C) Effects of upregulated MTX1 expression on cell proliferation were validated by the clonogenic assay. (D) . Forced expression of MTX1 increased xenograft tumor growth in nude mice. Tumor weights were measured after the mice were killed. Tumor volumes were recorded every week. All of results are shown as mean ± SD from at least three experiments, *P < 0.05, **P < 0.01.

Article Snippet: MTX1 antibody (15529-1-AP, Proteintech, Wuhan, China) was used to examine MTX1 expression in the HCC tissue microarray.

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

Journal: International Journal of Biological Sciences

Article Title: Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy

doi: 10.7150/ijbs.62393

Figure Lengend Snippet: MTX1 facilitated sorafenib resistance in HCC cells. (A) Cell growth was assessed by CCK-8 assay in PLC/PRF/5 (lv-VEC, lv-MTX1) and HCC-LM3 (lv-VEC, lv-MTX1) cells with or without sorafenib treatment. (B) Clonogenic capacity of cells with enhanced MTX1 expression was detected with or without sorafenib treatment. (C) Flow cytometry was used to analyze the effect of MTX1 expression on cell apoptosis induced by sorafenib or DMSO treatment. (D) The knockdown efficiency of siMTX1 in MTX1-overexpressed PLC/PRF/5 and HCC-LM3 cells by western blotting. (E) Cell growth was examined by CCK-8 assay when MTX1 was knocked down in MTX1 overexpressed HCC cells. All above data are mean ± SD from three experiments. * P<0.05, ** P<0.01.

Article Snippet: MTX1 antibody (15529-1-AP, Proteintech, Wuhan, China) was used to examine MTX1 expression in the HCC tissue microarray.

Techniques: CCK-8 Assay, Expressing, Flow Cytometry, Knockdown, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy

doi: 10.7150/ijbs.62393

Figure Lengend Snippet: MTX1 overexpression indicated anti-sorafenib effect in mouse model and HCC patients. (A) The mice were subcutaneously injected with Huh7 (lv-VEC, lv-MTX1) cells and treated with DMSO or sorafenib by oral gavage. The tumor picture and tumor weight were shown after mice were sacrificed. (B) Representative immunohistochemical images displayed different levels of MTX1 expression in human HCC tissues. Scale bars, up: 200 μm; down: 50 μm. (C) Kaplan-Meier analysis were performed to compare the difference of overall survival rates between HCC patients with low and high MTX1 expression (n = 80). Data are mean ± SD from three independent experiments. * P<0.05.

Article Snippet: MTX1 antibody (15529-1-AP, Proteintech, Wuhan, China) was used to examine MTX1 expression in the HCC tissue microarray.

Techniques: Over Expression, Injection, Immunohistochemical staining, Expressing

Journal: International Journal of Biological Sciences

Article Title: Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy

doi: 10.7150/ijbs.62393

Figure Lengend Snippet: MTX1 enhanced cell autophagy upon sorafenib treatment. (A) The relative expression levels of autophagy-related genes were detected by western blot in Huh7 cells with or without MTX1 overexpression. (B) Autophagosomes were viewed under transmission electron microscope in MTX1 overexpressed or control cells after sorafenib treatment. Red arrows point to autophagosomes. Scale bars: 1 μm. (C) Representative fluorescent images of mCherry-LC3B in HCC cells with or without MTX1 overexpression after sorafenib cultivation were displayed, and the number of LC3-positive puncta in each cell was counted. (D) Growth curves of HCC-LM3 cells under -/+ BafA1 conditions are shown. All cells in this assay were treated with 5 μM sorafenib. Quantitative data are presented as mean ± SD. NS: no significance, * P<0.05, ** P<0.01.

Article Snippet: MTX1 antibody (15529-1-AP, Proteintech, Wuhan, China) was used to examine MTX1 expression in the HCC tissue microarray.

Techniques: Expressing, Western Blot, Over Expression, Transmission Assay, Microscopy, Control

Journal: International Journal of Biological Sciences

Article Title: Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy

doi: 10.7150/ijbs.62393

Figure Lengend Snippet: MTX1 interacted with CISD1 to co-regulate HCC cell resistance to sorafenib. (A) Protein interaction networks were obtained by GeneMANIA analysis performed on Cytoscape (Red lines). (B) Co-IP and western blot assays were performed using anti-Flag and Dynabeads Protein G to confirm semi-exogenous interaction between FLAG-MTX1 and CISD1. (C) Endogenous CISD1 antibody could pull down MTX1-FLAG protein through Co-IP assay. (D, E) Autophagy-related proteins were detected by western blot analysis when CISD1 was silenced or overexpressed. (F) CISD1 overexpression antagonized MTX1 overexpression-induced autophagy-related protein expression under the treatment of sofafenib. (G) CISD1 reduced cell viability induced by MTX1 overexpression. CCK-8 assay was used to monitor cell viability at 0 and 48 h after the treatment of sofafenib in HCC cells as indicated. Data are presented as mean ± SD from three experiments. NS: no significance, * P<0.05, ** P<0.01.

Article Snippet: MTX1 antibody (15529-1-AP, Proteintech, Wuhan, China) was used to examine MTX1 expression in the HCC tissue microarray.

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

Journal: International Journal of Molecular Sciences

Article Title: Development of Drug Delivery Systems Based on Layered Hydroxides for Nanomedicine

doi: 10.3390/ijms15057750

Figure Lengend Snippet: Various applications of LHs.

Article Snippet: The MTX release from the nanocomposite at pH 7.4 occurred in two stages: at the beginning, around 50% of MTX was released in 7 h, followed by a relatively slower stage of 90% for the second 95 h and the release was completed in 190 h. The MTX release from the nanohybrids in pH 7.4 phosphate buffer saline followed relatively slow and fitting by the Ritger-Peppas model ( ) showed that the mass transfer process occurred through a combination of crystal dissolution, ion-exchange, and diffusion [ ].

Techniques: Adsorption, Synthesized, Concentration Assay, Generated, Activity Assay, Battery, Encapsulation, In Situ