Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet: Humanin-induced chemoresistance requires ATR signaling (A) hGBM1 cells were stimulated with HN or vehicle (Ctrl.), underwent transcriptomics, and differentially expressed genes (DEGs) were analyzed by bioinformatics. (B) Experiments described in (A) were repeated with hGBM-1, 2, and 3 cells providing 12 consistent DEGs, of which several components assembled in a network. (C) HUS1 was associated with outcome in human GBMs. (D) In a myeloid-free brain sample, hGBMs have a basal level of HUS1 expression, which is upregulated by interaction with hiPSC microglia in a GP130-dependent manner. (E and F) Contribution of the ATR pathway to humanin-induced GBM expansion (E) and chemoresistance (F) was demonstrated with the ATR inhibitor AZ20. (G) Western blots showing expression levels of HUS1, ATR and beta-actin (loading control) and a readout for of ATR activation (pT1989) in hGBM1 cells treated with bovine serum albumin (control), TMZ, HN, or AZ20. (H) In summary, AZ20 does not cooperate with TMZ per se, but blocks HN-induced TMZ resistance. The number of biological replicates is indicated (dots in graphs indicate data from individual experiments); all error bars are presented as mean ± SDM. Statistical significance is shown as FDR in (A), one-way ANOVA (D, E), or two-way ANOVA (F): ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; NS, not significant.

Article Snippet: HUS1 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-3364-pZIP-hCMV-ZsGreen-GVO-TRI.

Techniques: Expressing, Western Blot, Control, Activation Assay

Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet: Humanin-induced chemoresistance can be blocked therapeutically (A) Tumor size of orthotopic HN-WT or HN-C8a tumors was compared in mice receiving TMZ or vehicle (in animals with established tumor growth, 5x per week for 2 weeks; pre-defined endpoint was at 3 weeks). (B) Orthotopic hGBM1 was infused with HN (100 nM) or vehicle (artificial cerebrospinal fluid, aCSF) and i.p. injected with bazedoxifene-A (5 injections of BZA per week; 40 mg/kg; for 2 weeks) or vehicle; brains were labeled for HUS1; HUS1 expression was quantified. (C) Mice with established, orthotopic HN-WT tumors received TMZ (50 mg/kg) and were cotreated with vehicle or BZA (as in B); after 3 weeks, tumor size was quantified (dashed line: average data from untreated WT GBMs). (D) Mice with HN-WT GBMs received TMZ and were cotreated with vehicle or BZA (as in C); GBM samples were immunostained for active caspase-3 and immunolabeling was quantified (dashed line: average data from untreated WT GBMs). (E) Intratumoral vascularization and vessel diameter were compared in HN-WT or HN-C8a tumors receiving TMZ. (F) The HN-WT GBM mouse model was i.p. injected with TMZ and cotreated either with BZA or vehicle and the extent of intratumoral vascularization was compared. The number of biological replicates is indicated (dots in graphs indicate data from individual mice); all error bars are presented as mean ± SDM. Statistical significance is shown by one-way ANOVA (A, E), two-way ANOVA (B–D), or t test (F): ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; NS, not significant. Scale bars in (B, C) indicate 1 mm; scales in (D) represent 500 (overview) or 10 μm (magnified).

Article Snippet: HUS1 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-3364-pZIP-hCMV-ZsGreen-GVO-TRI.

Techniques: Injection, Labeling, Expressing, Immunolabeling

Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet:

Article Snippet: HUS1 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-3364-pZIP-hCMV-ZsGreen-GVO-TRI.

Techniques: Plasmid Preparation, Recombinant, Transfection, Fluorescence, Staining, Reverse Transcription, Expressing, Liposomes, Mutagenesis, shRNA, Control, Construct, Software, Imaging, Functional Assay, Dissection, Sequencing, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: lncRNA FEZF1-AS1 promotes migration, invasion and epithelial-mesenchymal transition of retinoblastoma cells by targeting miR-1236-3p

doi: 10.3892/mmr.2020.11478

Figure Lengend Snippet: Oligonucleotide sequences used for the transfection experiments.

Article Snippet: Synthetic sequences of short hairpin RNA (shRNA) targeting FEZF1-AS1 (shRNA-FEZF1-AS1; Shanghai GenePharma Co., Ltd.) and non-targeting shRNA (shRNA-NC) were inserted into pGPU6/Neo vector (Shanghai GenePharma Co., Ltd.).

Techniques: Transfection, Sequencing, shRNA

Journal: Molecular Medicine Reports

Article Title: lncRNA FEZF1-AS1 promotes migration, invasion and epithelial-mesenchymal transition of retinoblastoma cells by targeting miR-1236-3p

doi: 10.3892/mmr.2020.11478

Figure Lengend Snippet: Overexpression of FEZF1-AS1 in Rb cells. (A) Expression levels of FEZF1-AS1 were analyzed in four Rb cell lines and ARPE-19 cells (control) using RT-qPCR. **P<0.01, ***P<0.001 vs. control. (B) RT-qPCR analysis of the transfection efficiency of shRNA-FEZF1-AS1. ***P<0.001 vs. shRNA-NC group. All data are expressed as the mean ± SEM. Rb, retinoblastoma; FEZF1-AS1, FEZ family zinc finger 1 antisense RNA 1; RT-qPCR, reverse transcription- quantitative PCR; shRNA, short hairpin RNA; NC, negative control.

Article Snippet: Synthetic sequences of short hairpin RNA (shRNA) targeting FEZF1-AS1 (shRNA-FEZF1-AS1; Shanghai GenePharma Co., Ltd.) and non-targeting shRNA (shRNA-NC) were inserted into pGPU6/Neo vector (Shanghai GenePharma Co., Ltd.).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, shRNA, Real-time Polymerase Chain Reaction, Negative Control

Journal: Molecular Medicine Reports

Article Title: lncRNA FEZF1-AS1 promotes migration, invasion and epithelial-mesenchymal transition of retinoblastoma cells by targeting miR-1236-3p

doi: 10.3892/mmr.2020.11478

Figure Lengend Snippet: Silencing FEZF1-AS1 inhibits cell viability, migration and invasion. (A) Cell viability was determined in Y79 cells transfected with shRNA-NC or shRNA-FEZF1-AS1 using a Cell Counting Kit-8 assay. (B) Cell migratory rate was determined in Y79 cells transfected with shRNA-NC or shRNA-FEZF1-AS1 using a wound healing assay. Magnification, ×100. (C) Cell invasive rate was determined in Y79 cells transfected with shRNA-NC or shRNA-FEZF1-AS1 using a Transwell Matrigel invasion assay. Magnification ×100. All data are expressed as the mean ± SEM. ***P<0.001 vs. shRNA-NC group. FEZF1-AS1, FEZ family zinc finger 1 antisense RNA 1; shRNA, short hairpin RNA; NC, negative control.

Article Snippet: Synthetic sequences of short hairpin RNA (shRNA) targeting FEZF1-AS1 (shRNA-FEZF1-AS1; Shanghai GenePharma Co., Ltd.) and non-targeting shRNA (shRNA-NC) were inserted into pGPU6/Neo vector (Shanghai GenePharma Co., Ltd.).

Techniques: Migration, Transfection, shRNA, Cell Counting, Wound Healing Assay, Invasion Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: lncRNA FEZF1-AS1 promotes migration, invasion and epithelial-mesenchymal transition of retinoblastoma cells by targeting miR-1236-3p

doi: 10.3892/mmr.2020.11478

Figure Lengend Snippet: Epithelial-mesenchymal transition of retinoblastoma cells is suppressed by shRNA-FEZF1-AS1. Western blotting was used to analyze the protein expression levels of (A) Vimentin, Snail, Slug and β-catenin, (B) N-cadherin, E-cadherin, Claudin-1 and (C) MMP2 and MMP9 in Y79 cells transfected with shRNA-NC or shRNA-FEZF1-AS1. All data are expressed as the mean ± SEM. **P<0.01, ***P<0.001 vs. shRNA-NC group. FEZF1-AS1, FEZ family zinc finger 1 antisense RNA 1; shRNA, short hairpin RNA; NC, negative control; MMP, matrix metalloproteinase.

Article Snippet: Synthetic sequences of short hairpin RNA (shRNA) targeting FEZF1-AS1 (shRNA-FEZF1-AS1; Shanghai GenePharma Co., Ltd.) and non-targeting shRNA (shRNA-NC) were inserted into pGPU6/Neo vector (Shanghai GenePharma Co., Ltd.).

Techniques: shRNA, Western Blot, Expressing, Transfection, Negative Control

Journal: Molecular Medicine Reports

Article Title: lncRNA FEZF1-AS1 promotes migration, invasion and epithelial-mesenchymal transition of retinoblastoma cells by targeting miR-1236-3p

doi: 10.3892/mmr.2020.11478

Figure Lengend Snippet: miR-1236-3p is a direct target of FEZF1-AS1 in retinoblastoma cells. (A) Binding sites between FEZF1-AS1 and miR-1236-3p were predicted using LncBase v.2 software. (B) RNA immunoprecipitation assay was performed to analyze the interaction between miR-1236-3p and FEZF1-AS1 in Y79 cells co-transfected with miR-1236-3p mimic or miR-NC mimic and FEZF1-AS1-WT or FEZF1-AS1-MUT. ***P<0.001 vs. IgG group. (C) Relative luciferase activity was determined using a dual-luciferase reporter assay in cells co-transfected with miR-1236-3p mimic or miR-NC mimic and FEZF1-AS1-WT or FEZF1-AS1-MUT. ***P<0.001 vs. miR-NC mimic group. (D) Expression levels of miR-1236-3p were detected in Y79 cells transfected with shRNA-NC or shRNA-FEZF1-AS1 using RT-qPCR. ***P<0.001 vs. shRNA-NC group. (E) Expression levels of FEZF1-AS1 were analyzed in Y79 cells transfected with miR-NC mimic or miR-1236-3p mimic by RT-qPCR. **P<0.01 vs. miR-NC mimic group. All data are expressed as the mean ± SEM. FEZF1-AS1, FEZ family zinc finger 1 antisense RNA 1; shRNA, short hairpin RNA; NC, negative control; miR, microRNA; WT, wild-type; MUT, mutant; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: Synthetic sequences of short hairpin RNA (shRNA) targeting FEZF1-AS1 (shRNA-FEZF1-AS1; Shanghai GenePharma Co., Ltd.) and non-targeting shRNA (shRNA-NC) were inserted into pGPU6/Neo vector (Shanghai GenePharma Co., Ltd.).

Techniques: Binding Assay, Software, Immunoprecipitation, Transfection, Luciferase, Activity Assay, Reporter Assay, Expressing, shRNA, Quantitative RT-PCR, Negative Control, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: lncRNA FEZF1-AS1 promotes migration, invasion and epithelial-mesenchymal transition of retinoblastoma cells by targeting miR-1236-3p

doi: 10.3892/mmr.2020.11478

Figure Lengend Snippet: miR-1236-3p inhibitor reverses shRNA-FEZF1-AS1-induced suppression over cell viability, migration and invasion. (A) Cell viability was determined in Y79 cells transfected with shRNA-NC or shRNA-FEZF1-AS1 with or without miR-1236-3p inhibitor or miR-NC inhibitor using a Cell-Counting Kit-8 assay. (B and D) Migratory rates were determined in Y79 cells transfected with shRNA-NC or shRNA-FEZF1-AS1 with or without miR-1236-3p inhibitor or miR-NC inhibitor using wound healing assay. Magnification, ×100. (C and E) Invasive rates were determined in Y79 cells transfected with shRNA-NC or shRNA-FEZF1-AS1 with or without miR-1236-3p inhibitor or miR-NC inhibitor using Transwell Matrigel assay. Magnification, ×100. All data are expressed as the mean ± SEM. ***P<0.001 vs. shRNA-NC group; ## P<0.01 vs. shRNA-FEZF1-AS1 + miR-NC inhibitor group. FEZF1-AS1, FEZ family zinc finger 1 antisense RNA 1; shRNA, short hairpin RNA; NC, negative control; miR, microRNA.

Article Snippet: Synthetic sequences of short hairpin RNA (shRNA) targeting FEZF1-AS1 (shRNA-FEZF1-AS1; Shanghai GenePharma Co., Ltd.) and non-targeting shRNA (shRNA-NC) were inserted into pGPU6/Neo vector (Shanghai GenePharma Co., Ltd.).

Techniques: shRNA, Migration, Transfection, Cell Counting, Wound Healing Assay, Matrigel Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: lncRNA FEZF1-AS1 promotes migration, invasion and epithelial-mesenchymal transition of retinoblastoma cells by targeting miR-1236-3p

doi: 10.3892/mmr.2020.11478

Figure Lengend Snippet: Continued. Inhibition of miR-1236-3p reverses the effects of shRNA-FEZF1-AS1 on epithelial-mesenchymal transition. (A) Western blotting was used to analyze the protein expression levels of (A) Vimentin, Snail, Slug and β-catenin, (B) N-cadherin, E-cadherin and Claudin-1, and (C) MMP2 and MMP9 in Y79 cells transfected with shRNA-NC or shRNA-FEZF1-AS1 with or without miR-1236-3p inhibitor or miR-NC inhibitor. All data are expressed as the mean ± SEM. ***P<0.001 vs. shRNA-NC group; # P<0.05, ## P<0.01, ### P<0.001 vs. shRNA-FEZF1-AS1 + miR-NC inhibitor group. FEZF1-AS1, FEZ family zinc finger 1 antisense RNA 1; shRNA, short hairpin RNA; NC, negative control; miR, microRNA; MMP, matrix metalloproteinase.

Article Snippet: Synthetic sequences of short hairpin RNA (shRNA) targeting FEZF1-AS1 (shRNA-FEZF1-AS1; Shanghai GenePharma Co., Ltd.) and non-targeting shRNA (shRNA-NC) were inserted into pGPU6/Neo vector (Shanghai GenePharma Co., Ltd.).

Techniques: Inhibition, shRNA, Western Blot, Expressing, Transfection, Negative Control

Journal: BMC Cancer

Article Title: Interaction between DNMT3B and MYH11 via hypermethylation regulates gastric cancer progression

doi: 10.1186/s12885-021-08653-3

Figure Lengend Snippet: Primer used for RT-qPCR

Article Snippet: The overexpressed (oe) DNA plasmids oe-MYH11, oe-TNFRSF14, oe-DNMT3B and control oe-negative control (NC) used for cell transfection were from VectorBuilder (Guangzhou, Guangdong, China).

Techniques:

Journal: BMC Cancer

Article Title: Interaction between DNMT3B and MYH11 via hypermethylation regulates gastric cancer progression

doi: 10.1186/s12885-021-08653-3

Figure Lengend Snippet: DNMT3B upregulation is responsible for the hypermethylation of the MYH11 promoter. A , the expression of DNMT3A and DNMT3B in GC was queried in StarBase Pan-cancer platform; B , the enrichment ability of DNMT3A and DNMT3B on MYH11 promoter examined by ChIP-qPCR; C , DNMT3B mRNA expression in GC tumor tissues and their adjacent tissues by RT-qPCR; D , correlation of DNMT3B expression with MYH11 promoter methylation levels in tumor tissues analyzed by Pearson’s correlation analysis (r = 0.623, p < 0.001); E , correlation of DNMT3B expression with MYH11 expression in tumor tissues (r = − 0.609, p < 0.001); F , transfection efficiency of oe-DNMT3B in GC cells by RT-qPCR; G , the effects of DNMT3B overexpression on the methylation level of MYH11 promoter examined by qMSP; H , effects of DNMT3B overexpression on MYH11 expression by RT-qPCR. Each assay was performed at least three times. Statistical significance was analyzed by paired t test (panel C) or two-way ANOVA (panels B, F, G and H) and Tukey’s multiple range tests

Article Snippet: The overexpressed (oe) DNA plasmids oe-MYH11, oe-TNFRSF14, oe-DNMT3B and control oe-negative control (NC) used for cell transfection were from VectorBuilder (Guangzhou, Guangdong, China).

Techniques: Expressing, Quantitative RT-PCR, Methylation, Transfection, Over Expression

Journal: BMC Cancer

Article Title: Interaction between DNMT3B and MYH11 via hypermethylation regulates gastric cancer progression

doi: 10.1186/s12885-021-08653-3

Figure Lengend Snippet: Mechanism diagram. Overexpression of DNMT3B in GC inhibited MYH11 expression by promoting methylation of the MYH11 promoter, thereby attenuating the repressive effect of MYH11 on TNFRSF14 transcriptional activity and promoting GC progression

Article Snippet: The overexpressed (oe) DNA plasmids oe-MYH11, oe-TNFRSF14, oe-DNMT3B and control oe-negative control (NC) used for cell transfection were from VectorBuilder (Guangzhou, Guangdong, China).

Techniques: Over Expression, Expressing, Methylation, Activity Assay

Journal: Helicobacter

Article Title: HIF ‐1α‐Induced GPR171 Expression Mediates CCL2 Secretion by Mast Cells to Promote Gastric Inflammation During Helicobacter pylori Infection

doi: 10.1111/hel.70042

Figure Lengend Snippet: GPR171 partially mediates CCL2 production via the ERK1/2 pathway. (A–C) GPR171 expression was verified in GPR171‐knockdown RBL‐2H3 cells by RT‐qPCR and western blotting. (D) Percentage of β‐hexosaminidase released into the supernatants from control shRNA‐treated and GPR171 shRNA‐treated RBL‐2H3 cells in response to H. pylori infection. (E) The mRNA levels of the 6 cytokines in control shRNA‐treated and GPR171 shRNA‐treated cells stimulated with H. pylori . (F) CCL2 levels in the supernatants of shRNA‐treated and GPR171 shRNA‐treated RBL‐2H3 cells co‐cultured with H. pylori . (G) GO enrichment of DEGs based on RNA‐seq data. (H) Immunoblot analysis and quantification of ERK1/2 phosphorylation in RBL‐2H3 cells that were or were not treated with H. pylori . p‐ERK expression was normalized to total ERK. (I) RT‐qPCR analysis of CCL2 mRNA expression in mast cells co‐cultured with or without H. pylori . (J) CCL2 levels in supernatants were quantified by ELISA. Cells were pretreated with 5 μM U0126 as indicated (H–J). (K) Immunoblot analysis and quantification of p‐ERK in control shRNA‐treated and GPR171 shRNA‐treated cells following H. pylori stimulation. (L, M) Western blot analysis of ERK1/2 phosphorylation expression in RBL‐2H3 cells treated with different concentrations of MS21570 (0, 5, 10, and 20 μM) and co‐cultured with or without H. pylori . (N, O) RT‐qPCR and ELISA analysis of CCL2 expression in RBL‐2H3 cells treated with different concentrations of MS21570 (0, 5, 10, and 20 μM) and co‐cultured with or without H. pylori . MS21570: A blocker of GPR171. Statistical significance was determined by Student's unpaired t ‐test (A, C, D, E, F, K) and one‐way ANOVA (H, I, J, M, N, O), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Lentiviral vectors carrying GFP and either shRNA targeting GPR171 mRNA or a non‐targeting control shRNA were customized (GenePharma, China).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Control, shRNA, Infection, Cell Culture, RNA Sequencing, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: VCP/p97-governed proteostasis is tied to metabolic pathways and nutrient availability. a Network graph of leading-edge enriched genes from the top three most highly enriched KEGG pathways. Edge weight and size indicate strength of correlation between genes/nodes. Nodes are coloured by the pathways each gene contributes to. b GSEA plots of the KEGG term Metabolic Pathways, with genes ranked by correlation with VCP/p97 expression, in the Cancer Cell Line Encyclopedia (CCLE) and in patient-derived tumour cells in the Myeloma IX study. The correlation value of individual genes is indicated by black bars (left axis) and running enrichment score is highlighted by red line (right axis). NES normalised enrichment score. c Relative mRNA levels of the indicated genes in A549 cells stably expressing non-targeting control (NTC) or one of two IPTG-inducible VCP/p97 shRNAs after 48 h of IPTG treatment in complete medium (25 mM glucose, 2 mM glutamine), followed by 24 h maintenance in complete, low glucose (1 mM glucose, 2 mM glutamine) or no glutamine (25 mM glucose, 0 mM glutamine) medium supplemented with 500 μM IPTG. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). d Relative mRNA levels of the indicated genes in A549 cells grown under different conditions of nutrient availability and treated with CB5083 (1 μM) as indicated for 16 h. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). e Proportion of non-viable A549 cells as determined by Annexin V and 7-AAD staining after treatment with CB5083 under the indicated conditions of nutrient availability for 48 h. Data shown are mean and SEM, n = 3 (two-way ANOVA with Sidak’s multiple comparisons test). f VCP/p97 mRNA expression in the indicated cell lines grown in complete, low glucose or no glutamine medium for 16 h (A549), 24 h (DU145), or 48 h (A172). Data shown are mean and SEM, n = 3 (Mann–Whitney test)

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Expressing, Derivative Assay, Stable Transfection, Control, Staining, MANN-WHITNEY

Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: Metabolite responses to VCP/p97 inhibition. A549 cells were grown in complete (25 mM glucose, 2 mM glutamine), low glucose (1 mM glucose, 2 mM glutamine), or no glutamine (25 mM glucose, 0 mM glutamine) medium for 16 h followed by intracellular metabolite extraction and gas chromatography-mass spectrometry. Data show the effects of CB5083 treatment (1 μM) for 16 h. a Volcano plots indicating metabolites with significant changes (coloured). The horizontal dashed line represents a multiple testing-adjusted p -value of 0.05, and the vertical dashed lines represent log 2 fold changes of 0.67 and 1.5, respectively. b Bar graphs show the effect of treatment with CB5083 on individual metabolites (fold change of treatment to control for each of the three media compositions). Comparisons with significant effects (two-way ANOVA, n = 4 per experimental condition) are outlined in red

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Inhibition, Extraction, Gas Chromatography, Mass Spectrometry, Control

Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: GCN2 modulates proteotoxic stress and ERK signalling in response to VCP/p97 inhibition. a Immunoblot for GCN2 and β-tubulin on extracts from untransduced A549 cells and A549 cells stably expressing targeting (shGCN2) or non-targeting (shNTC) shRNA . b-e A549 cells stably expressing non-targeting control (shNTC) or one of two shRNAs targeting GCN2 (shGCN2) were grown in complete (25 mM glucose, 2 mM l -glutamine), low glucose (1 mM glucose, 2 mM glutamine), or no glutamine (25 mM glucose, 0 mM glutamine) medium. CB5083 was used at 1 μM. b Proportion of non-viable A549 cells as determined by Annexin V-FITC and 7-AAD staining after 48 h of experimental treatment. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). c mRNA levels relative to shNTC expressing cells grown in complete medium after 16 h of experimental treatment. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). d Immunoblot analyses on whole-cell extracts from A549 cells (three independent experiments) stably expressing shNTC or shGCN2(1). Bar graphs show relative phosphorylated ERK1/2 immunoblot band intensities (mean and SEM) compared to control cells. e Proportion of non-viable A549 cells as determined by Annexin V and 7-AAD staining after 24 h of experimental treatment with AZD6244, U0126, and CB5083 for 24 h. Mean and SEM of n = 3 experiments is shown (two-way ANOVA and Tukey’s multiple comparisons for effects of AZD6244 and U0126 compared to vehicle-treated or CB5083-treated cells; two-way ANOVA and Bonferroni’s multiple comparisons test for effects of shGCN2 compared to shNTC under the same experimental conditions (grey asterisks); exact p values for all comparisons are shown in Supplementary Figure )

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Inhibition, Western Blot, Stable Transfection, Expressing, shRNA, Control, Staining

Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: GCN2 attenuates autophagy in response to metabolic and proteotoxic stress. A549 cells stably expressing non-targeting control (shNTC) or one of two shRNAs targeting GCN2 (shGCN2) were grown in complete (25 mM glucose, 2 mM l -glutamine), low glucose (1 mM glucose, 2 mM glutamine), or no glutamine (25 mM glucose, 0 mM glutamine) medium. CB5083 was used at 1 μM. a GSEA plot of KEGG autophagy pathway with genes ranked by correlation with VCP/p97 expression. The correlation value of individual genes is indicated by black bars (left axis) and running enrichment score is highlighted by red line (right axis). NES normalised enrichment score. b mRNA levels relative to shNTC expressing cells grown in complete medium after 16 h of experimental treatment. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). c Immunoblot analyses on whole-cell extracts from A549 cells (three independent experiments) stably expressing shNTC or shGCN2(1). Bar graphs show relative LC3BII and p62 immunoblot band intensities, compared to control cells (mean and SEM). d Immunoblot analyses on whole-cell extracts from A549 cells treated with CB5083 (1 μM) for 24 h and bafilomycin A1 (100 nM) for 16 h. e Propoof non-viable A549 cells treated with CB5083 (1 μM) for 48 h and bafilomycin A1 (100 nM) for 40 h. Data shown are mean and SEM, n = 3 (two-way ANOVA and Tukey’s multiple comparisons test)

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Stable Transfection, Expressing, Control, Western Blot

Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: Effects of GCN2 on metabolites and protein synthesis. a Bar graphs show metabolite levels in shGCN2 A549 cells compared to shNTC cells in different media (fold change for each of the three media compositions (16 h), and metabolite levels in shGCN2(1) cells (fold change to shNTC cells) after treatment with CB5083 (1 μM, 16 h). Comparisons with significant effects are outlined in red ( n = 4, two-way ANOVA). b , c Immunoblot analyses for the indicated proteins on whole-cell extracts from A549 cells (three independent experiments) stably expressing shNTC or shGCN2(1). Bar graphs show relative immunoblot band intensities, compared to control cells (mean and SEM)

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Western Blot, Stable Transfection, Expressing, Control

Journal: Journal of Translational Medicine

Article Title: Artemisinin conferred cytoprotection to human retinal pigment epithelial cells exposed to amiodarone-induced oxidative insult by activating the CaMKK2/AMPK/Nrf2 pathway

doi: 10.1186/s12967-024-05593-x

Figure Lengend Snippet: The Nrf2 pathway was involved in the cytoprotective effect of artemisinin on amiodarone-induced cell death in D407 cell cultures. ( a-b ) The cells were treated with artemisinin at various concentrations for 1 h. The Nrf2 and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( c ) The cell cultures were incubated with or without the Nrf2 inhibitor ML385 at concentrations ranging from 2.5 to 15 µM for 24 h, and cell viability was measured using MTT ( n = 4). ( d-e ) Cells attached to 12-well plates were treated with 5 or 10 µM ML385 for 24 h. The Nrf2 and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 4). ( f-g ) The cell cultures were pretreated with 5 µM ML385 for 6, 12, or 24 h, followed by 20 µM artemisinin for 1 h and 5 µM amiodarone for another 24 h. Cell viability was determined by the MTT assay ( n = 4), and cell death was detected through the LDH cytotoxicity assay ( n = 4). ( h-j ) Cells attached to 24-well plates were transfected with control shRNA (shCTL) or Nrf2 shRNA (shNrf2) plasmids (0.5 µg/well) for 72 h. The Nrf2, HO-1, and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( k ) Cells transfected with shCTL or shNrf2 plasmids were attached to 96-well plates, and pretreated with artemisinin for 1 h, then treated with amiodarone for another 24 h and processed for LDH assay ( n = 4)

Article Snippet: The human AMPKɑ2 shRNA plasmids (target sequence: TGTGAAAGAAGTGTGTGAA), control shRNA plasmids (CON207, target sequence: TTCTCCGAACGTGTCACGT), the AMPKɑ2-overexpressing plasmids, and control overexpressing plasmids (CON238) were purchased from GenePharma Co., Ltd. (Shanghai, China).

Techniques: Western Blot, Incubation, MTT Assay, LDH Cytotoxicity Assay, Transfection, Control, shRNA, Lactate Dehydrogenase Assay

Journal: Journal of Translational Medicine

Article Title: Artemisinin conferred cytoprotection to human retinal pigment epithelial cells exposed to amiodarone-induced oxidative insult by activating the CaMKK2/AMPK/Nrf2 pathway

doi: 10.1186/s12967-024-05593-x

Figure Lengend Snippet: AMPK-mediated activation of the Nrf2/HO-1 pathway was linked to the cytoprotective effect of artemisinin on amiodarone-induced apoptosis in D407 cell cultures. ( a-d ) The cell cultures were pretreated with 1 µM STO-609 for 30 min or 1 µM Compound C for 30 min, followed by 20 µM artemisinin for 1 h and 5 µM amiodarone for another 24 h. The HO-1, cleaved caspase-3, and GAPDH protein levels were detected by western blotting, and the protein bands were quantified by Image J ( n = 3). ( e-h ) Cells attached to 12-well plates were transfected with shCTL or AMPKɑ2 shRNA (shAMPKɑ2) plasmids (2 µg/well) for 48 h. The AMPKɑ2, HO-1, cleaved caspase-3, and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( i-j ) Cells attached to 24-well plates were transfected with the AMPKɑ2-overexpressing (OE-AMPKɑ2) or control-overexpressing (OE-CTL) plasmids (0.5 µg/well) for 24 h. The AMPKɑ2 and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( k ) The cell cultures transfected with OE-CTL or OE-AMPKɑ2 plasmids for 24 h were attached to 96-well plates and treated with 5 µM amiodarone for 24 h, and processed for the LDH assay ( n = 4)

Article Snippet: The human AMPKɑ2 shRNA plasmids (target sequence: TGTGAAAGAAGTGTGTGAA), control shRNA plasmids (CON207, target sequence: TTCTCCGAACGTGTCACGT), the AMPKɑ2-overexpressing plasmids, and control overexpressing plasmids (CON238) were purchased from GenePharma Co., Ltd. (Shanghai, China).

Techniques: Activation Assay, Western Blot, Transfection, shRNA, Control, Lactate Dehydrogenase Assay