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: 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: Nature neuroscience

Article Title: Kcnn2 blockade reverses learning deficits in the mouse model of Fetal Alcohol Spectrum Disorders

doi: 10.1038/s41593-020-0592-z

Figure Lengend Snippet: a ) Timeline of the experiment. ( b ) Representative image of a brain that received Kcnn2 (or control) knockdown (visualized by co-expressed GFP) in the motor area (outlined by red rectangles). ( c ) Immunohistochemistry for Kcnn2 (red) shows that Kcnn2 shRNA, but not control shRNA, suppresses the increase in Kcnn2 expression in layer III neurons in M1 in PAE mice. Arrowheads indicate Kcnn2 + cells among GFP + electroporated cells. ( d ) Percentage of Kcnn2 + cells among GFP + electroporated cells in the indicated experimental groups. **P = 0.001 by two-tailed Student’s t -test (n = 10 per group). ( e , f ) Motor learning deficits in PAE mice, revealed by lower success rate ( e ) and learning index ( f ) in the single pellet reaching test, are mitigated by Kcnn2 knockdown in layer II/III neurons in M1. A significant interaction was observed between the effects of condition (treatment plus shRNA) and trial ( e ); F(1,11) = 2.80, P = 0.01 by two-way repeated measures ANOVA, *P < 0.05, **P < 0.01 by simple main effect test [PAE (Kcnn2 shRNA − ) vs PAE (Kcnn2 shRNA + )], and between treatment and shRNA ( f ); F(1,24) = 5.55, P = 0.03 by two-way ANOVA, **P < 0.05, ***P < 0.005 by simple main effect test (n = 10 animals per group). Graph shows mean ± SEM. In box plots ( d , f ), the line within the box indicates the median, and the upper and lower edges of the box represent the 25th and 75th percentiles, respectively. The upper and lower whisker boundaries indicate the 10th and 90th percentiles, respectively, and dots indicate outliers.

Article Snippet: The control non-targeting shRNA was purchased (SuperArray Bioscience Corporation, MD).

Techniques: Control, Knockdown, Immunohistochemistry, shRNA, Expressing, Two Tailed Test, Whisker Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: The relative expression of BCAT1 in Ishikawa and HEC-1A cells. BCAT1 expression in Ishikawa and HEC-1A cells transfected with a blank control, shCtrl and shBCAT1. The y-axis indicates the relative expression of BCAT1 in each group tested using RT-qPCR.

Article Snippet: Short Hairpin RNA (shRNA) - mediated BCAT1 knockdown in HEC-1A and Ishikawa cells To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: A. Heat map showing the changes in the concentrations (Z-score) of 26 amino acids and their derivatives after BCAT1 knockdown in HEC-1A cells. B. Intracellular amino acids and their derivative levels in shCtrl cells and shBCAT1 cells.

Article Snippet: Short Hairpin RNA (shRNA) - mediated BCAT1 knockdown in HEC-1A and Ishikawa cells To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Knockdown

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: CD109 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-135228-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: Heliyon

Article Title: Clinical significance of immune-related antigen CD58 in gliomas and analysis of its potential core related gene clusters

doi: 10.1016/j.heliyon.2024.e29275

Figure Lengend Snippet: shRNA interference cells screening and confirmation of transfection effect. (A) and (B) displayed qPCR and Western blot detection of CD58 expression in HEB, U251, and U87, respectively. (C) Images of cells after shRNA interference. Bar represented 100 μm. (D) CD58 protein expression in different shRNA interference groups. Results of qPCR and Western blot showed that U251 and U87 cells were suitable for shRNA interference. The fluorescence staining results showed successful transfection. Compared to the blank transfection group (NC), the CD58 expression was significantly downregulated in the CD5 8 shRNA transfection group (KD).

Article Snippet: CD5 8 shRNA (KD) and non-target control (NC) shRNA were synthesized by BGI Genomics (BGI Genomics Co., Ltd., China).

Techniques: shRNA, Transfection, Western Blot, Expressing, Fluorescence, Staining