Journal: Aging

Article Title: Age-related defects in autophagy alter the secretion of paracrine factors from bone marrow mononuclear cells.

doi: 10.18632/aging.203127

Figure Lengend Snippet: Figure 3. Atg7 knockdown in Y-Sca-1+ BMCs blocks paracrine stimulation of old cardiac fibroblasts. (A) Atg7 expression was measured in Y-Sca-1+ bone marrow cells (BMCs), treated with Atg7-siRNA or sc-siRNA, by RT-qPCR. n=3-4. (B–E) Quantification and representative images from (B) scratch wound assay, (C) proliferation assay, (D) stress fiber formation or (E) β-galactosidase staining of old cardiac fibroblasts, treated with Y-Sca-1+ Atg7-siRNA CM or Y-Sca-1+ sc-siRNA CM for 24-48 hours. Dashed yellow line in (B) indicates the wound edge at 0 hours. Scale bar represents 100 μm. Data analysis was carried out by t-test. Data presented as mean ± SEM; n=4-6; **p≤0.01.

Article Snippet: The nonspecific siRNA oligonucleotides (scrambled siRNA) and siRNA oligonucleotides targeting mouse Atg7 were purchased from OriGene (#SR427399). siRNA was transfected into Y-Sca-1+ BMCs using Lipofectamine 2000 (#11668-027, Invitrogen) according to the manufacturer’s protocol.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Scratch Wound Assay Assay, Proliferation Assay, Staining

Journal: Theranostics

Article Title: Midkine signaling maintains the self-renewal and tumorigenic capacity of glioma initiating cells

doi: 10.7150/thno.41450

Figure Lengend Snippet: MDK regulates the self-renewal capacity and tumorigenic properties of GICs. (A) MDK protein levels (as determined by ELISA) in the medium of GICs cultures or of their corresponding serum-differentiated cells. Data correspond to MDK concentration (ng/ml) and are expressed as mean concentration ± SEM (n=3). * P < 0.05; ** P < 0.01 from serum-differentiated cells. (B) Effect of MDK genetic inhibition [by treating with doxycycline (+Dox) cells stably transduced with a doxycycline-inducible MDK-selective (shMDK) shRNA] on the morphology of GH2, 12O12 and HCO1-GICs cultures. Representative images obtained by phase-contrast microscopy are shown. Values in each image correspond to the neurosphere's area (nm 2 ) and are expressed as mean fold change ± SEM. At least 4-5 neurospheres were analyzed for each experimental condition in each GIC culture. * P < 0.05 from shMDK (-Dox). (C) Effect of MDK genetic inhibition or incubation with an anti-MDK antibody (MDK Ab., 40 μg/ml) on the growth of GH2-GICs (n=3). ** P < 0.01 from vehicle or shMDK (-Dox) cells. (D) Effect of MDK genetic inhibition (+Dox) on the self-renewal ability (as determined by LDA) of GH2, 12O12 and HCO1-GICs (n=2). * P < 0.05; *** P < 0.001 from shMDK (-Dox). (E-F) Effect of MDK depletion by incubation with an anti-MDK antibody (MDK Ab., 40 μg/ml, 72h) (GH2-GICs, panel E) or by expressing a doxycycline-inducible shMDK (+Dox; HCO1-GICs, panel F) on the mRNA levels of a panel of stem cell associated genes. Data are expressed as mean fold change from vehicle (panel E) or shMDK (-Dox) (panel F)-treated cells (reference) ± SEM (n=3). * P < 0.05; ** P < 0.01; *** P < 0.001 from vehicle-treated cells (panel E) or from shMDK (-Dox) (panel F). (G) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 μg/ml, 72 h) on NESTIN expression (as determined by immunostaining) of 12O12-GICs. Scale bar: 20 μm. (H) Procedure to generate intracranial xenografts with 12O12 shMDK-GICs. (I) Effect of MDK genetic inhibition [shMDK (+Dox)] on the size of glioma xenografts generated by intracranial injection of 7.5 x 10 4 12O12 shMDK-GICs (n=12). Representative MRI images 6 weeks after the injection are shown (left panel). Tumor volume is expressed as mean ± SEM (right panel). * P < 0.05 from 12O12 shMDK (-Dox) tumors. (J) Effect of MDK genetic inhibition [shMDK (+Dox)] on the survival of tumor-bearing mice. Kaplan-Meier plot (n=12). *** P < 0.001 from 12O12 shMDK (-Dox) tumors.

Article Snippet: To stably knock-down ATG7 we used a SMART-pool of concentrated transduction- ready human lentiviral particles containing 3 target- specific constitutive shRNA constructs (sc-41447-V, Santa Cruz Biotechnology).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Inhibition, Stable Transfection, Transduction, shRNA, Microscopy, Incubation, Expressing, Immunostaining, Generated, Injection

Journal: Theranostics

Article Title: Midkine signaling maintains the self-renewal and tumorigenic capacity of glioma initiating cells

doi: 10.7150/thno.41450

Figure Lengend Snippet: MDK/ALK axis plays a relevant role in the maintenance of the stem-like properties and the tumorigenic capacity of GICs. (A) Effect of the incubation with exogenous MDK (MDK, 50 ng/ml) on the phosphorylation of ALK (pALK), AKT (pAKT), ERK (pERK) and STAT3 (pSTAT3) of GH2-GICs at different time points. Left panel: a representative Western blot is shown (n=3). Right panel: Data correspond to the densitometric analysis of the levels of each phosphorylated protein relative to total levels of that protein and are expressed as the mean fold change ± SEM relative to vehicle-treated cells (n=3). * P < 0.05 and *** P < 0.001 from MDK 0 min-treated cells. (B) Effect of MDK genetic inhibition [by treating with doxycycline (+Dox) cells stably transduced with a doxycycline-inducible MDK-selective (shMDK) shRNA] on the phosphorylation of ALK (pALK), AKT (pAKT), ERK (pERK), and STAT3 (pSTAT3) of GH2-GICs. Left panel: a representative Western blot is shown (n=3). Right panel: data correspond to the densitometric analysis of the levels of each phosphorylated protein relative to total levels of that protein and are expressed as the mean fold change ± SEM relative to shMDK (-Dox)-treated cells (n=3). *** P < 0.001 from (-Dox)-treated cells. (C) Effect of ALK genetic inhibition (by treating with doxycycline (+Dox) cells stably transduced with a doxycycline-inducible shALK) or incubation with TAE (1 μM) or crizotinib (0.75 μM) on the growth of GH2-GICs (n=3). *** P < 0.001 from vehicle or shALK (-Dox) cells. (D) Effect of ALK genetic inhibition (+Dox) or incubation with TAE (0.75 μM) on the self-renewal ability (as determined by LDA) of GH2, 12O12 and HCO1-GICs (n=2). ** P < 0.01 from shALK (-Dox.) cells or HCO1 vehicle-treated cells; *** P < 0.001 from GH2 vehicle-treated cells. (E) Effect of ALK genetic inhibition (by treating with doxycycline (+Dox) cells stably transduced with a doxycycline-inducible shALK) on the mRNA levels (as determined by qPCR) of a panel of stem cell associated genes in 12O12 shALK-GICs. Data are expressed as mean fold change from shALK (-Dox)-treated cells (reference) ± SEM (n=3). * P < 0.05; ** P < 0.01; *** P < 0.001 from 12O12shALK (-Dox). (F) Effect of the incubation with TAE (0.75 µM, 72 h) on NESTIN expression (as determined by immunofluorescence) of HCO1-GICs. Values in each photomicrograph correspond to the percentage of NESTIN-positive cells relative to the total number of nuclei. Representative photomicrographs are shown (n=3). *** P < 0.001 from vehicle-treated cells. Scale bar: 20 μm. (G) Effect of ALK genetic inhibition on the size of glioma xenografts (MRI) generated by intracranial injection of 7.5 x 10 4 12O12 shALK-GICs. Representative MRI images 6 weeks after the injection are shown (left panel). Tumor volume is expressed as mean ± SEM (right panel) (n=8-11). ** P < 0.01 from 12O12 shALK (-Dox) tumors. (H) Effect of ALK genetic inhibition on the survival of tumor-bearing mice. Kaplan-Meier plot (n=12). *** P < 0.001 from 12O12 shALK (-Dox) tumors. Inset: Effect of doxycycline (+Dox) on ALK protein levels of 12O12 shALK cultures determined right before their intracranial injection.

Article Snippet: To stably knock-down ATG7 we used a SMART-pool of concentrated transduction- ready human lentiviral particles containing 3 target- specific constitutive shRNA constructs (sc-41447-V, Santa Cruz Biotechnology).

Techniques: Incubation, Phospho-proteomics, Western Blot, Inhibition, Stable Transfection, Transduction, shRNA, Expressing, Immunofluorescence, Generated, Injection

Journal: Theranostics

Article Title: Midkine signaling maintains the self-renewal and tumorigenic capacity of glioma initiating cells

doi: 10.7150/thno.41450

Figure Lengend Snippet: MDK/ALK signaling axis regulates the maintenance of the stem-like properties of GICs by controlling SOX9 protein levels. (A) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 μg/ml) or TAE (0.75 μM) for 24 h on SOX2, SOX4, SOX9 and CD133 protein levels of GH2-GICs. Left panel: a representative Western blot experiment is shown (n=5). Right panel: densitometric analysis of SOX9, SOX2 and SOX4 protein levels (mean fold change from vehicle ± SEM; n=5 for SOX9 and SOX2 and n=3 for SOX4). ** P < 0.01 and *** P < 0.001 from vehicle-treated cells. NS: statistically non-significant differences. (B) Effect of SOX9 genetic inhibition [by nucleofection with a plasmid encoding a shcontrol (shC) or a SOX9-selective (shSOX9) shRNA] on the self-renewal ability (as determined by LDA) of GH2-GICs (n=3) ** P < 0.01 from shC cells. (C) Effect of SOX9 genetic inhibition (72 h) on mRNA levels of a panel of stem cell associated genes (as determined by qPCR) of GH2-GICs. Data are expressed as mean fold change from shC cells (reference) ± SEM (n=3). * P < 0.05; ** P < 0.01; ** P < 0.001 from shC cells. (D) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 μg/ml) and nucleofection with a control plasmid (CP), or a plasmid encoding a murine SOX9 (SOX9) on the self-renewal ability (as determined by LDA) of GH2-GICs (n=2). # P < 0.05 from MDK Ab.-treated CP cells. Full χ 2 statistical analysis is included in LDA statistics section within the . (E) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 µg/ml) on the mRNA levels of NESTIN , MUSASHI-1 (MSI1) and SOX9 (as determined by qPCR) of GH2-GICs nucleofected with a control plasmid (CP) or a plasmid encoding a murine SOX9 (SOX9) (72 h). * P < 0.05 from vehicle CP-treated cells.

Article Snippet: To stably knock-down ATG7 we used a SMART-pool of concentrated transduction- ready human lentiviral particles containing 3 target- specific constitutive shRNA constructs (sc-41447-V, Santa Cruz Biotechnology).

Techniques: Incubation, Western Blot, Inhibition, Plasmid Preparation, shRNA, Control

Journal: Molecular Neurobiology

Article Title: Amorfrutin B Protects Mouse Brain Neurons from Hypoxia/Ischemia by Inhibiting Apoptosis and Autophagy Processes Through Gene Methylation- and miRNA-Dependent Regulation

doi: 10.1007/s12035-022-03087-9

Figure Lengend Snippet: Silencing of Becn1 and/or Atg7 reduced the neuroprotective effects of amorfrutin B against hypoxia- and ischemia-induced cell damage. In Atg7 siRNA-transfected cells, amorfrutin B lost its neuroprotective capacity under hypoxic ( a ) but not ischemic ( b ) conditions. The results were normalized to the absorbance in negative control (scrambled siRNA). The results are presented as a percentage of the control ± SEM of 3 independent experiments, consisting of 8–12 replicates per group. *** p < 0.001 compared to normoxia, ### p < 0.001 compared to the cultures exposed to hypoxia, ^^^ p < 0.001 compared to the cultures exposed to ischemia, $$$ p < 0.001 compared to negative control

Article Snippet: The Becn1 and Atg7 siRNAs were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Transfection, Negative Control, Control