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: bioRxiv

Article Title: DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.1101/865626

Figure Lengend Snippet: (A) Schematic of DECR1 function in fatty acid (FA) β-oxidation. In order to translocate FAs into the mitochondria, CPT1 converts long-chain acyl-CoA species to their corresponding long-chain acylcarnitine species. This is followed by a dehydrogenation step mediated by acyl CoA dehydrogenase (ACAD) to generate trans-2-enoyl-CoA, the only intermediate that can be processed by downstream enzymes in the β-oxidation process. Many FAs have unsaturated bonds either on an odd-numbered carbon or in the cis-configuration, resulting in the generation of enoyl-CoA intermediates that cannot be directly processed via the downstream β-oxidation enzymes. These FAs require the activity of 3 auxiliary enzymes, ECI1, ECH1 and DECR1 in order to form trans-2-enoyl-CoA before undergoing β-oxidation. DECR1 catalyzes the conversion of either 2-trans,4-cis-dienoyl or 2-trans,4-trans-dienoyl-CoA to 3-trans-enoyl-CoA. A complete cycle of β-oxidation results in the release of the first two carbon units as acetyl-CoA, and a fatty-acyl-CoA minus two carbons. The acetyl-CoA enters the TCA cycle to produce energy (ATP). The shortened fatty-acyl-CoA is processed again starting with the ACADs to form trans-2-enoyl-CoA either directly or with the aid of the auxiliary enzymes depending on the presence of double bonds. This process continues until all carbons in the fatty acid chain are turned into acetyl-CoA. (B) DECR1 protein expression after 72 hours or 96 hours siRNA transfection. Densitometry quantification of relative DECR1 protein expression was normalized to the HSP90 internal control. (C) Linoleic acid level in LNCaP cells quantified in following 96 hours DECR1 knockdown using GC QQQ targeted metabolomics. (D) Relative quantities of the C10:2 acylcarnitine species in LNCaP cell conditioned medium (left) or cell lysates (right) (n=3). (E) Quantification of ATP levels in LNCaP cell lysates. LNCaP cells were transfected with DECR1 siRNAs for 48 hours and then starved in no-glucose medium and treated with the lipolysis inhibitor DEUP (100µM) in the presence (BSA-LA) or absence (BSA) of the PUFA linoleic acid for 48 hours before measuring ATP levels. ( F) Oxygen consumption rate (OCR) was assessed in LNCaP cells supplemented with the PUFA linoleic acid (LA) or (G) the saturated fatty acid palmitic acid (PA). Each data point represents an OCR measurement. ATP production, maximal mitochondrial respiration and mitochondrial spare capacity were assessed. (H) Extracellular acidification rate (ECAR) was assessed in LNCaP cells. Each data point represents an ECAR measurement. For experiments (F-H) LNCaP cells were transfected with DECR1 siRNAs for 72 hours, then starved in substrate limited medium for 24 hours; the assay was run in FAO assay medium. (I & J) Metabolites were quantified in LNCaP cells following 96 hours DECR1 knockdown using GC QQQ targeted metabolomics. Data in bar graphs are represented as the mean ± s.e.m (n=3). Statistical analysis was performed using two-tailed Student’s t -test: * p <0.05, ** p <0.01 and **** p <0.0001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc. (San Diego, CA, USA) according to the manufacturer’s protocol.

Techniques: Activity Assay, Expressing, Transfection, Control, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.1101/865626

Figure Lengend Snippet: (A) Cell viability after DECR1 knockdown in non-malignant PNT1 prostate cells; hormone-responsive PCa cell lines (LNCaP and VCaP); castrate-resistant V16D and 22RV1 cell lines and enzalutamide-resistant MR94F cells cultured in full serum media. (B) Cell viability of stable DECR1-overexpressed LNCaP cells cultured in full serum media. Cell viability and cell death were measured using trypan blue exclusion following 96 hours DECR1 knockdown. Percentages are represented relative to the control siRNA; n = 3 independent experiments per cell line. (C) Clonogenic cell survival of LNCaP cells were assessed using colony formation assay. Stable DECR1-overexpressed cells or (D) stable DECR1 knockdown was achieved using two different short hairpin (sh) vectors and DECR1 expression was confirmed using western blot. Cells were cultured for 2 weeks, washed with PBS, fixed with paraformaldehyde and stained with 1% crystal violet for 30 minutes. Colonies with more than 50 cells were counted manually; data shown is representative of n = 2 independent experiments. (E) LNCaP and 22RV1 cell growth in 3D spheres. Spheroids were prepared using the hang drop assay following 48 hours DECR1 knockdown. Spheroid volumes were determined after five days of culturing the cells in 20 µl drops; at least 25 spheres per cell line were assessed using the ReViSP software, n = 3 independent experiments per cell line. (F) LNCaP, 22RV1 and MR49F cell migration and (G) 22RV1 cell invasion were assessed using transwell migration/invasion assay. Cells were transfected with DECR1 siRNA or control siRNA for 48 hours. Equal number of cells were transferred to the upper inserts in serum free medium; lower chambers were filled with medium containing 5% serum as a chemoattractant. Plates were incubated for a further 48 hours. Migrated/invaded cells on the lower face of the inserts were washed with PBS, fixed with paraformaldehyde, stained with 1% crystal violet for 30 minutes, and counted manually; data shown is representative of n = 3 independent experiments. (H) Violin plots of mKi67 and DECR1 mRNA expression in LNCaP tumors (n = 5 mice, shControl; n = 4 mice, shDECR1). (I) Representative KI67 IHC staining of LNCaP tumors. Scale bar, 100µm. Data in bar graphs are represented as the mean ± s.e.m. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test: * p <0.05, ** p <0.01, *** p <0.001 and **** p <0.0001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc. (San Diego, CA, USA) according to the manufacturer’s protocol.

Techniques: Knockdown, Cell Culture, Control, Colony Assay, Expressing, Western Blot, Staining, Software, Migration, Invasion Assay, Transfection, Incubation, Immunohistochemistry

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