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: Scientific Reports

Article Title: Efficient nucleic acid delivery to murine regulatory T cells by gold nanoparticle conjugates

doi: 10.1038/srep28709

Figure Lengend Snippet: Freshly isolated eGFP + regulatory T cells were cultured in presence or absence of 1.6 × 10 6 or 3.2 × 10 6 siRNA-coupled nanoparticle conjugates (5 nm) per cell or ligand-free particles as controls. eGFP expression was analyzed as mean fluorescence intensity (MFI) on gated living cells by flow cytometry ( A ) at day 2 or ( B ) day 3 and calculated as percentage of CD25 + untreated (= 100%). Results from three independent experiments are summarized as mean ± SEM. One-way ANOVA with Bonferroni´s post test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Successful transfection of T lymphocytes with plasmids or siRNA contructs was also achieved by others using transient plasmonic nanobubble injection or optimized electroporation protocols based on the Lonza nucleofection technique.

Techniques: Isolation, Cell Culture, Expressing, Fluorescence, Flow Cytometry

Journal: American Journal of Physiology - Cell Physiology

Article Title: Abundance of TRPC6 protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes

doi: 10.1152/ajpcell.00014.2011

Figure Lengend Snippet: Changes in the planar surface area of MCs in response to ANG II. A and B: representative photomicrographs of MCs used in contraction assays. A: MCs were cultured in NG (5.6 mM d-glucose + 20 mM l-glucose) and were transfected with either enhanced green fluorescent protein (EGFP) alone or EGFP plus shRNA construct specific for human TRPC6 (EGFP + hTRPC6-shRNA). B: MCs were cultured in HG (25.6 mM d-glucose) and were transfected with either EGFP alone or EGFP tagged TRPC6 expression plasmid (TRPC6-EGFP). The green cells indicate the positively transfected MCs and were used for analysis indicated by arrows. C: summary data from the experiments presented in A (Ca) and B (Cb) showing the contractile response of MCs with different treatments, calculated as [(the surface area of MCs after ANG II − the surface area of MCs before ANG II)/the surface area of MCs before ANG II] × 100%. n indicates the number of cells analyzed in each group. *P < 0.05; **P < 0.01 compared with EGFP group.

Article Snippet: As described by Du et al. ( 7 ) with modifications, human MCs were plated on a 22 × 22 mm coverslip and were transiently transfected with either green fluorescent protein (GFP) + hTRPC6-short hairpin RNA (shRNA) constructs (1:10, GFP:hTRPC6-shRNA) or TRPC6-enhanced GFP (EGFP) constructs using GenJet (SignaGen, Gaithersburg, MD).

Techniques: Cell Culture, Transfection, shRNA, Construct, Expressing, Plasmid Preparation