small interfering rnas against atg5  (Santa Cruz Biotechnology)

Journal: Journal of neuroinflammation

Article Title: TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy.

doi: 10.1186/s12974-023-02959-8

Figure Lengend Snippet: Fig. 3 TRIM45 regulates autophagy proteins associated with NLRP3. a qRT-PCR analysis of p62, Atg5, and beclin1 mRNA levels in BV2 cells; n = 3 per group. b The autophagy proteins p62, beclin1, Atg5 and LC3 in BV2 cells were measured by western blotting. c Changes in ROS in BV2 cells in the different groups; n = 3 per group. d JC-1 staining to detect mitochondrial membrane potential and cellular morphology was examined by a phase-contrast microscope. (scale bar = 75 μm); n ≥ 3 per group. Data in a, c, d were analysed by one-way ANOVA followed by Dunnett’s post hoc test. Data are presented as the mean ± S.E.M. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Article Snippet: Small interfering RNAs against Atg5 (sc-41446, Santa Cruz) were purchased from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Western Blot, Staining, Membrane, Microscopy

Journal: Journal of neuroinflammation

Article Title: TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy.

doi: 10.1186/s12974-023-02959-8

Figure Lengend Snippet: Fig. 4 TRIM45 regulates Atg5 based on its E3 ubiquitin ligase activity. a Western blot analysis of Atg5 expression in BV2 cells in response to different interventions. b BV2 cells with TRIM45 knockdown or control cells were treated with cycloheximide (CHX) for 0, 2 and 4 h. c BV2 cells were immunoprecipitated with IgG and anti-Atg5 antibodies, and the expression of TRIM45 and Atg5 was detected by western blotting. d Protein confidence analysis of Atg5 and TRIM45 was performed by high-throughput interactive group (TRIM45 (green), Atg5 (blue)). e BV2 cells were transfected with HA-TRIM45 for 48 h. BV2 cells were immunoprecipitated with an anti-Atg5 antibody and then subjected to western blot analysis with an anti-NLRP3 antibody. f Co-IP analysis of Atg5 ubiquitination in BV2 cells. The cell extracts were collected for IP with anti-Atg5 beads, followed by IP analysis with the indicated antibodies. g HEK293T cells were transfected with TRIM45 and HA-tagged ubiquitin plasmids with or without LPS + ATP treatment. The cell extracts were collected for IP with anti-Atg5 beads, followed by IP analysis with the indicated antibodies. h, i HEK293T cells were transfected with plasmids TRIM45 and HA-tagged k63 or k48 ubiquitin with or without LPS + ATP treatment. The cell extracts were collected for IP with anti-Atg5 beads, followed by IP analysis with the indicated antibodies

Article Snippet: Small interfering RNAs against Atg5 (sc-41446, Santa Cruz) were purchased from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Activity Assay, Western Blot, Expressing, Knockdown, Control, Immunoprecipitation, High Throughput Screening Assay, Transfection, Co-Immunoprecipitation Assay

Journal: Journal of neuroinflammation

Article Title: TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy.

doi: 10.1186/s12974-023-02959-8

Figure Lengend Snippet: Fig. 5 TRIM45 regulates the NLRP3 pathway in an Atg5-dependent manner. a, b Western blot analysis and quantification of NLRP3, Gsdmd-N, Atg5 and IL-1β levels in BV2 cells transfected with siTRIM45 or HA-TRIM45 and stimulated by LPS + ATP; n = 3 per group. c BV2 cells were infected with siTRIM45 and Flag-Atg5; n = 4 per group. d BV2 cells were infected with HA-TRIM45 and siAtg5; n = 4 per group. e, f Statistical analysis of Figures c and d. g, h Flow cytometry was used to analyse BV2 pyroptosis; n = 3 per group. Data in a, b, h were analysed by one-way ANOVA followed by Dunnett’s post hoc test. Data in c, d were analysed by unpaired t test. Data are presented as the mean ± S.E.M. from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Article Snippet: Small interfering RNAs against Atg5 (sc-41446, Santa Cruz) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Transfection, Infection, Flow Cytometry

Journal: Science advances

Article Title: Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.

doi: 10.1126/sciadv.adf9284

Figure Lengend Snippet: Fig. 1. Autophagy inhibition leads to a drop in the LIP in PDAC. (A) Autophagy was inhibited genetically or pharmacologically in PDAC cells, and the relative LIP was determined after cotreatment with FAC in 8988T cells. (B) Chloroquine (CQ)– mediated drop in LIP was rescued by FAC in the indicated PDAC cell lines. (C) OCR rescue after FAC cotreatment in CQ-treated or ATG5-knockdown PDAC cells. Heat- maps showing clonogenic assays (D) in PDAC cells and relative proliferation in a panel of PDAC cell lines after treatment with FAC in autophagy-inhibited cells. Data are means ± SD, and P values were quantified using two-way analysis of variance (ANOVA) with Sidak’s multiple comparison’s test (for A, B, D, and E) and one-way ANOVA with Tukey’s post hoc test (for C). **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered as significant. The heatmaps (A, B, D, and E) as well as OCR data of siATG5 panel (C) are representative of one experiment repeated n = 3 (for heatmaps) and 2 (for OCR) times, respectively, while the OCR data of CQ experiment panel in (C) are combined data from n = 3 experiments. Heatmaps are indicating values in % by considering control as 100.

Article Snippet: Small interfering RNA (siRNA) pools against ATG5 (sc-41445), LC3 (sc-43390), FTH (sc-40575), FTL (sc-40577), NCOA4 (sc-29719), SLC4A7 (sc77885), and IL-6 (sc-39627) were purchased from Santa Cruz Biotechnology Inc., along with control siRNA (sc-37007), which consisted of a scrambled sequence.

Techniques: Inhibition, Knockdown, Control

Journal: Science advances

Article Title: Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.

doi: 10.1126/sciadv.adf9284

Figure Lengend Snippet: Fig. 3. Loss of autophagy impairs mitochondrial microarchitecture in PDAC. (A) Representative TEM images of PDAC cells treated with PBS (Control) or CQ followed by determining their (B) mitochondrial number per cell (Control, n = 14; CQ, n = 12 cells). (C) A magnified section of a PDAC cell showing mitochondrial ultrastructure after CQ treatment followed by quantification of (D) crista number per mitochondria (n = 17 unique crista for both Control and CQ), (E) crista lumen width (Control, n = 18; CQ, n = 13 unique crista), and (F) crista length (n = 19 unique crista for both Control and CQ). (G) Representative TEM images of ATG5-knockdown PDAC cells with or without FAC treatment were quantified for (H) crista length and (I) crista lumen width (siControl, n = 13; siATG5, n = 9; siATG5 + FAC, n = 11 unique crista). Black boxes were digitally zoomed in to highlight the morphology of typical dysfunctional mitochondria. Random TEM image of mitochondria was blindly acquired and quantified. Data are means ± SD, and P values were quantified using one-way ANOVA with Tukey’s post hoc test. **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered as significant.

Article Snippet: Small interfering RNA (siRNA) pools against ATG5 (sc-41445), LC3 (sc-43390), FTH (sc-40575), FTL (sc-40577), NCOA4 (sc-29719), SLC4A7 (sc77885), and IL-6 (sc-39627) were purchased from Santa Cruz Biotechnology Inc., along with control siRNA (sc-37007), which consisted of a scrambled sequence.

Techniques: Control, Knockdown

Journal: Science advances

Article Title: Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.

doi: 10.1126/sciadv.adf9284

Figure Lengend Snippet: Fig. 4. Ectopic SDHB rescues the mitochondrial dysfunction upon autophagy inhibition in PDAC. PDAC cells expressing SDHB and control vector were treated with CQ followed by immunoblotting for the indicated proteins (A) and analyzed for (B) OCR (combined data from n = 2 experiments). (C) Representative TEM images of cells are shown in situation similar to (A) followed by mitochondrial crista length (D; n = 18 unique cristae for all groups) and lumen width (E; n = 18 unique cristae for all groups) determination. Red arrows in (C) indicate the typical dysfunctional mitochondria. ATG5 was suppressed in SDHB overexpression and control PDAC cells for immunoblotting of indicated proteins (F). Under conditions similar to (F), cell proliferation (G; combined data of n = 5 experiment) and OCR was determined (H; combined data of n = 2 experiments). Data are means ± SD, and P values were quantified using one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered as significant.

Article Snippet: Small interfering RNA (siRNA) pools against ATG5 (sc-41445), LC3 (sc-43390), FTH (sc-40575), FTL (sc-40577), NCOA4 (sc-29719), SLC4A7 (sc77885), and IL-6 (sc-39627) were purchased from Santa Cruz Biotechnology Inc., along with control siRNA (sc-37007), which consisted of a scrambled sequence.

Techniques: Inhibition, Expressing, Control, Plasmid Preparation, Western Blot, Over Expression

Journal: Science advances

Article Title: Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.

doi: 10.1126/sciadv.adf9284

Figure Lengend Snippet: Fig. 6. ISCA1 overexpression rescues the mitochondrial architecture changes after autophagy inhibition. (A) Representative TEM images of PDAC cells overex- pressing ISCA1 or control vector after CQ treatment was done followed by analysis of mitochondrial crista length (B) and lumen width (C) from n = 12 unique cristae for all groups. Red arrows in (A) indicate the typical dysfunctional mitochondria. (D) Effect of ISCA1 expression on the OCR was estimated after CQ treatment (representative data from one experiment that was repeated n = 2 times). (E) ATG5 was suppressed in cells bearing ISCA1 overexpression, and the level of indicated proteins was measured by immunoblotting, followed by measurement of relative cell proliferation (F; combined data of n = 5 experiments) and OCR (G; representative data of one experiment that was repeated n = 2 times). Data are means ± SD, and P values were quantified using one-way ANOVAwith Tukey’s post hoc test. **P < 0.01, ***P < 0.001, and ****P < 0.0001 *P < 0.05 were considered as significant.

Article Snippet: Small interfering RNA (siRNA) pools against ATG5 (sc-41445), LC3 (sc-43390), FTH (sc-40575), FTL (sc-40577), NCOA4 (sc-29719), SLC4A7 (sc77885), and IL-6 (sc-39627) were purchased from Santa Cruz Biotechnology Inc., along with control siRNA (sc-37007), which consisted of a scrambled sequence.

Techniques: Over Expression, Inhibition, Control, Plasmid Preparation, Expressing, Western Blot

Journal: Science advances

Article Title: Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.

doi: 10.1126/sciadv.adf9284

Figure Lengend Snippet: Fig. 8. CAFs compensate for the LIP deficit in autophagy-inhibited PDAC. (A) PDAC cells transfected with siRNAs against ATG5 were cocultured with CAFs followed by quantification of LIP in PDAC. (B) Mono- or cocultured PDAC and CAFs under conditions similar to (A) and assessed for the indicated proteins by immunoblotting. (C) Cell culture supernatants of PDAC cells treated with CQ or expressing siATG5 were analyzed using the proteome profiler human cytokine array kit (combined data from n = 2 repeats), and the change in cytokine pixel intensity was quantified in (D). (E) Under conditions similar to (C), IL-6 was quantified by enzyme-linked immunosorbent assay (representative data from one experiment repeated n = 3 times). Data are means ± SD. For (A), (E), and (D), P values were quantified using one-way (Tukey’s post hoc test) and two-way ANOVA (Holm Sidak’s test), respectively. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered as significant.

Article Snippet: Small interfering RNA (siRNA) pools against ATG5 (sc-41445), LC3 (sc-43390), FTH (sc-40575), FTL (sc-40577), NCOA4 (sc-29719), SLC4A7 (sc77885), and IL-6 (sc-39627) were purchased from Santa Cruz Biotechnology Inc., along with control siRNA (sc-37007), which consisted of a scrambled sequence.

Techniques: Transfection, Western Blot, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells

doi: 10.1186/s12967-017-1223-7

Figure Lengend Snippet: Inhibition of necroptosis does not affect shikonin-induced apoptosis. a Cells were transfected with control siRNA or RIP siRNA. After 6 h of transfection, the cells were incubated with 3 or 6 µM shikonin for 3 h before analysis by western blotting. The cell lysates were subjected to 10 or 15% SDS-PAGE to measure the expression of the indicated proteins. Immunoblots are representative of at least two independent experiments. b Necroptosis was evaluated by annexin V/propidium iodide staining. c Values are presented as mean ± SD of three independent experiments. * p < 0.01 compared with the shikonin-treated group

Article Snippet: Pooled small interfering RNA (siRNA) oligonucleotides against ATG5 were purchased from Cell Signaling Technology. siRNA against RIP1 was purchased from Ambion (Austin, TX, USA).

Techniques: Inhibition, Transfection, Control, Incubation, Western Blot, SDS Page, Expressing, Staining

Journal: Journal of Translational Medicine

Article Title: Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells

doi: 10.1186/s12967-017-1223-7

Figure Lengend Snippet: Inhibition of autophagy increases shikonin-induced necroptosis. Cells were treated with 3 or 6 µM shikonin in the presence or absence of autophagy inhibitors, i.e., 1 mM 3-MA ( a ), 50 nM bafilomycin A ( d ), and ATG5 siRNA ( g ). The cell lysates were subjected to 10 and 15% SDS-PAGE to measure the expression of indicated proteins. b , e , h Necroptosis was evaluated by annexin V/propidium iodide. c , f , i Values are presented as mean ± SD from three independent experiments. * p < 0.01 compared with the shikonin group

Article Snippet: Pooled small interfering RNA (siRNA) oligonucleotides against ATG5 were purchased from Cell Signaling Technology. siRNA against RIP1 was purchased from Ambion (Austin, TX, USA).

Techniques: Inhibition, SDS Page, Expressing