human atg5 shrna plasmids  (Genechem)

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Autophagy-dependent sensitization effects of PARP inhibitors on recurrent nasopharyngeal carcinoma treated with carbon ion and photon irradiation

doi: 10.3724/abbs.2025130

Figure Lengend Snippet: Autophagy inhibition attenuates the radiation-sensitizing effect of olaparib (A) HK-1 cells were plated in 96-well plates and treated with X-ray or C-ion irradiation, with or without olaparib (5 μM), in the presence of different cell death inhibitors. Z-VAD (15 μM), Nec-1 (15 μM) and Fer-1 (5 μM) were added 24 h before radiation exposure, and 3-MA (5 μM) was added 2 h before radiation. All the inhibitors were incubated for 24 h postirradiation. The morphology of the cells was observed and imaged 48 h after irradiation via an inverted microscope (20× magnification; scale bar: 100 μm). (B,C) HK-1 cells were treated as described in (A). Left panel: Cell survival was assessed via CCK-8 assay 48 h after X-ray or C-ion exposure, with the values normalized to those of the nonirradiated control of the corresponding drug group. Right panel: The proportion of surviving cells rescued by each inhibitor in the X-ray + olaparib or C-ion + olaparib combination treatment group was calculated and is presented as a pie chart. The “other” category represents additional cell death mechanisms, determined as one minus the sum of the cell survival fractions rescued by the four inhibitors. (D) The gene knockdown efficiency of three ATG5-targeting shRNAs was confirmed via western blot analysis. GAPDH served as the loading control. Sh-ATG5-67 clones were selected for subsequent studies. (E) Cells transfected with sh-ATG5 or sh-NC were treated with olaparib and/or radiation, and their autophagy levels were measured by flow cytometry 48 h after irradiation. (F,G) ATG5 knockdown significantly increased the HK-1 cell survival rate following X-ray or C-ion irradiation, with or without olaparib (5 μM). The numbers in parentheses indicate the number of cells plated per well for each dose group. Survival curves were generated for the X-ray (H) and C-ion (I) groups, with the values normalized to those of the nonirradiated group. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: For ATG5 knockdown, three human ATG5 shRNA plasmids constructed by GeneChem were used: shATG5-65: 5′-GATTCATGGAATTGAGCCAAT-3′; shATG5-66: 5′-CCTTTCATTCAGAAGCTGTTT-3′; shATG5-67: 5′-CCTGAACAGAATCATCCTTAA-3′.

Techniques: Inhibition, Irradiation, Incubation, Inverted Microscopy, CCK-8 Assay, Control, Knockdown, Western Blot, Clone Assay, Transfection, Flow Cytometry, Generated

Journal: Autophagy

Article Title: Aberrant SIRT6 expression contributes to melanoma growth: Role of the autophagy paradox and IGF-AKT signaling

doi: 10.1080/15548627.2017.1384886

Figure Lengend Snippet: The bimodal role of SIRT6 in melanoma growth at different stages in vivo. (A) Primary and metastatic melanoma cells stably transfected with SIRT6 overexpression vectors or SIRT6 shRNA vectors were subcutaneously injected into NOD/SCID nude mice (n = 5 per group) for the generation of subcutaneous xenograft tumors. Tumor volumes including tumor length (L) and width (W) were measured using vernier calipers every 3 d from d 6 after injection and then calculated according to the formula (L × W2)/2. Data are presented as means ± SD (from 5 individual mice). **, P < 0.01, FLAG and Ctr-shRNA as control, respectively. (B) Tumor weights were analyzed at the terminal time point. At the end of 33 d, tumors were excised and weighed. The data are shown as means ± SD (from 5 mice per group). **, P < 0.01, FLAG and Ctr-shRNA as control, respectively. (C and D) Tumors from sacrificed mice were dissected 33 d after subcutaneous injection and are shown in the indicated row representatively (n = 4 per group). (E and F) Representative immunoblotting analysis showing the expressions of IGF-AKT signaling-related proteins and autophagy markers (LC3 and SQSTM1) in tumors from sacrificed mice as indicated (n = 3 per group). WT1 and WT2, SIRT6-WT1 and SIRT6-WT2; FLAG, FLAG-tagged control vector; shRNA1 and shRNA2, SIRT6-shRNA1 and SIRT6-shRNA2; Ctr-shRNA, control shRNA. N, FLAG or Ctr-shRNA; W and T, SIRT6-WT1 and SIRT6-WT2; S and H, SIRT6-shRNA1 and SIRT6-shRNA2.

Article Snippet: Retroviral vectors synthesizing ATG5 shRNA or control shRNA were purchased from Ori Gene (TR314610).

Techniques: In Vivo, Stable Transfection, Transfection, Over Expression, shRNA, Injection, Western Blot, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: ATG Ubiquitination Is Required for Circumsporozoite Protein to Subvert Host Innate Immunity Against Rodent Malaria Liver Stage

doi: 10.3389/fimmu.2022.815936

Figure Lengend Snippet: CSP inhibits the IFN-γ-mediated suppression of EEFs by the downregulation of ATGs. (A) 1.2 × 10 5 control and CSP-stably transfected HepG2 cells were infected with Ad-mRFP-GFP-LC3 virus, and then treated with the autophagy inducer rapamycin for 24 h. Both LC3-RFP or LC3-GFP puncta in HepG2 cells were imaged ( up ) and qualified ( down ). Scale bar = 5 μm, n=60. (B) 1.2 × 10 5 control and CSP-stably transfected HepG2 cells were transiently transfected with LC3-RFP plasmid, and then incubated with 4 × 10 4 CSP wt or CSP mut sporozoites for 24 h. Cells were fixed and stained with anti-UIS4, and LC3 surrounding the EEFs was observed under confocal microscopy ( up ), and the intensity of LC3 around PV was quantified ( down ), Scale bar = 5 μm, n (EEFs) =60. (C) HepG2 (without lentivirus infection), control (infected with Lenti-pCDH control lentivirus and selected by puromycin) and CSP-stably transfected HepG2 cells were treated with rapamycin for the indicated times. Protein levels of ATGs, including LC3I/LC3II, ATG3, ATG5, ATG7, Beclin-1, and p62 (SQSTM1), was determined by western blotting. (D) Control and CSP-stably transfected HepG2 cells transfected with or without ATG5, or ATG7 plasmid, and then treated with 1 U/mL IFN-γ and infected with CSP wt sporozoites as above for 46 h. The parasite load in HepG2 cells was determined and compared. Three individual experiments have been performed. (E) HepG2 cells were transiently transfected with Scramble or ATG5 shRNA plasmids, and then treated with 1 U/mL IFN-γ and incubated with CSP wt or CSP mut sporozoites as above. 46 h later, the parasite load in the HepaG2 cells was determined and compared. The experiment has been performed for three times. (F) Both control (ATG5 fl/fl ) and ATG5 fl/fl - Alb - Cre mice were infected by intravenous injection with 1,000 CSP wt or CSP mut sporozoites, 46 h later, the liver burden of the two parasites in control and ATG5 fl/fl - Alb - Cre mice were determined and compared. Each dot represents one mouse, n=15. The pooled data of three repeated experiments was presented, data are represented as mean ± SD, and analyzed by a student’s t-test, Mann-Whitney U test or One-Way ANOVA; ns, not significant; **P < 0.01; ***P < 0.001.

Article Snippet: RNA interference for ATG5 and ATG7 in HepG2 cells was performed according to the protocol of the ATG5/7 human shRNA plasmid kit (Origene, Rockville, MD, USA).

Techniques: Stable Transfection, Transfection, Infection, Plasmid Preparation, Incubation, Staining, Confocal Microscopy, Western Blot, shRNA, Injection, MANN-WHITNEY