Journal: International journal of biological sciences

Article Title: Radiosensitizer EXO-miR-197-3p Inhibits Nasopharyngeal Carcinoma Progression and Radioresistance by Regulating the AKT/mTOR Axis and HSPA5-mediated Autophagy.

doi: 10.7150/ijbs.69934

Figure Lengend Snippet: Figure 6. MiR-197-3p downregulating HSPA5 modulates autophagy to control X-ray sensitivity. (A) The expression of autophagy related proteins in transfected cells was detected by Western Blot. (B, C) The changes of autophagosomes after transfection with GFP-LC3 were detected. (D) Western Blot experiments verified that miR-197-3p regulated autophagy by targeting HSPA5. (E) The expression of HSPA5 and autophagy related protein LC3B in transfected cells was detected by cell immunofluorescence. (F) Immunofluorescence showed that tumors infected with LV-miR-197-3p expressed HSPA5 and LC3B in animal tissue. (G) Western Blot experiment showed the expression of LC3BI/LC3BII and HSPA5 in animal tumor protein. (H) Immunohistochemical results showed the expression of HSPA5, Ki-67, BCL2, LC3B and P62.

Article Snippet: The used antibodies are as follows: GAPDH (Transgen, HC301-01), HSPA5 (Proteintect, 11587-1-AP), LC3B (Abcam, ab192890), P62 (BOSTER, M00300-1), Phospho-mTOR (Ser2448) (D9C2) (Cell Signaling Technology, 5536T), mTOR (7C10) (Cell Signaling Technology, 2983T), PhosphoAkt (Ser473) (D9E) (Cell Signaling Technology, 4060S), Akt (pan) (C67E7) (Cell Signaling Technology, 4691S), Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (ab275018), Goat Anti-mouse IgG (Transgen, HS201-01), Goat anti-rabbit IgG (Transgen, HS101-01).

Techniques: Control, Expressing, Transfection, Western Blot, Immunofluorescence, Infection, Immunohistochemical staining

Journal: International journal of biological sciences

Article Title: Radiosensitizer EXO-miR-197-3p Inhibits Nasopharyngeal Carcinoma Progression and Radioresistance by Regulating the AKT/mTOR Axis and HSPA5-mediated Autophagy.

doi: 10.7150/ijbs.69934

Figure Lengend Snippet: Figure 8. EXO-miR-197-3p regulates radioresistance and autophagy by targeting HSPA5. (A, B) Colony formation assay was detected the radioresistance of the cells ingested EXO-miR-197-3p. (C, D, E, F) Flow cytometry was detected apoptosis after uptake of exosomes at 4GY. (G) Western Blot was detected the expression of HSPA5 after cell uptake of EXO-miR-197-3p. (H) Animal experiments tested the effect of EXO-miR-197-3p. (I, J) Tumor volume and weight were measured. (K) Immunohistochemical results showed that after EXOs-miR-197-3p was injected; the expressions of HSPA5, Ki-67, BCL2, LC3B and P62 were analyzed. (L) Immunohistofluorescence showed that the expression of HSPA5 and LC3B; Immunohistofluorescence showed that the expression of HSPA5 and LC3B was decreased in the EXO group. (M) Western Blot assay detected the expression of LC3B and HSPA5 in tumor. (N) Western Blot detected the expression of HSPA5 after cell uptake of EXO-miR-197-3p. (O, P) After transfection GFP-LC3, autophagosome formation was detected after EXO-HK-1 ingestion. (Q) The expressions of HSPA5 and autophagy related protein LC3B in cell uptake of EXO-HK-1 was detected by cell immunofluorescence. (R) The correlation between miR-197-3p, HSPA5 and LC3B was detected by Western Blot.)

Article Snippet: The used antibodies are as follows: GAPDH (Transgen, HC301-01), HSPA5 (Proteintect, 11587-1-AP), LC3B (Abcam, ab192890), P62 (BOSTER, M00300-1), Phospho-mTOR (Ser2448) (D9C2) (Cell Signaling Technology, 5536T), mTOR (7C10) (Cell Signaling Technology, 2983T), PhosphoAkt (Ser473) (D9E) (Cell Signaling Technology, 4060S), Akt (pan) (C67E7) (Cell Signaling Technology, 4691S), Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (ab275018), Goat Anti-mouse IgG (Transgen, HS201-01), Goat anti-rabbit IgG (Transgen, HS101-01).

Techniques: Colony Assay, Flow Cytometry, Western Blot, Expressing, Immunohistochemical staining, Injection, Immunohistofluorescence, Transfection, Immunofluorescence

Journal: PLoS ONE

Article Title: Neuroprotective Effect of TAT-14-3-3ε Fusion Protein against Cerebral Ischemia/Reperfusion Injury in Rats

doi: 10.1371/journal.pone.0093334

Figure Lengend Snippet: (A) Representative electron microphotographs showing autophagosomes in the ischemic penumbra of cerebral cortex of sham, vehicle or TAT-14-3-3ε pre-treated animals 24 h after reperfusion. Autophagosomes are indicated by arrows. Scale bar = 1 μm. (B) Western blot analysis for the expression of LC3B, p62 and Beclin-1 in cerebral hemisphere. (C, D and E) Quantitation of LC3B-II, p62 and Beclin-1 expression, respectively, normalized to β-actin. The data are presented as the mean ± SD (n = 5 per group). # p <0.05 and ## p <0.01 versus sham group. * p <0.05 versus vehicle group.

Article Snippet: Polyclonal rabbit anti-LC3B antibody (1∶1000, Santa Cruz, CA, USA), polyclonal rabbit anti-Beclin-1 antibody (1∶1000, Santa Cruz, CA, USA) and polyclonal rabbit anti-p62/SQSTSM1 antibody (1∶500, Abgent, CA, USA) were used for immunoreactivity detection.

Techniques: Western Blot, Expressing, Quantitation Assay

Journal: PLoS ONE

Article Title: Neuroprotective Effect of TAT-14-3-3ε Fusion Protein against Cerebral Ischemia/Reperfusion Injury in Rats

doi: 10.1371/journal.pone.0093334

Figure Lengend Snippet: Rats were treated with an i.c.v of saline or 3-MA (600 nmol) at the end of 2h ischemia, or RAP (35 pmol, i.c.v.) combined with TAT-14-3-3ε (10 mg/kg, i.v.) 2 h before 2 h MCAO. Rats were then subjected to 24 h reperfusion, after which all animals were sacrificed. (A) Western blot analysis of the LC3B, p62 and Beclin-1 protein expression in the ischemic cerebral hemisphere. (B, C and D) Quantitation of LC3B-II, p62 and Beclin-1 expression, respectively, normalized to the loading control (β-actin). Bar represents mean ± SD (n = 4 per group). ## p <0.01 compared with sham group. * p <0.05 compared with vehicle group. § p <0.05 compared with RAP + TAT-14-3-3ε group.

Article Snippet: Polyclonal rabbit anti-LC3B antibody (1∶1000, Santa Cruz, CA, USA), polyclonal rabbit anti-Beclin-1 antibody (1∶1000, Santa Cruz, CA, USA) and polyclonal rabbit anti-p62/SQSTSM1 antibody (1∶500, Abgent, CA, USA) were used for immunoreactivity detection.

Techniques: Western Blot, Expressing, Quantitation Assay

Journal: Toxins

Article Title: Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells

doi: 10.3390/toxins13100684

Figure Lengend Snippet: Western blot analysis of quinoin-treated primary cell lines for 24 h. ( A ) Quinoin induced a significant time-independent reduction of Cyclin D1 and ( B ) activation of apoptosis by a decrease of procaspase 3 when administered at a concentration of 250 nM in the NULU cell line. ( C ) Western blot analysis of the expression of Cyclin D1, ( D ) procaspase, and autophagic markers p62 ( E ) and LC3B ( F ) after treatment of the ZAR cell line with different concentrations of quinoin for 24 h. Densitometric analysis of protein levels represent the means ± SEM of three individual determinations. Data were normalized to the housekeeping gene actin and are expressed as a fold change over control-treated cells. * Unpaired t -test. According to GraphPad Prism, * p -value 0.01 to 0.05 (significant), ** p -value 0.001 to 0.01 (very significant), *** p -value 0.0001 to 0.001 (extremely significant).

Article Snippet: To monitor the expression of the cell cycle protein Cyclin D1, the apoptotic protein procaspase 3, and the autophagic proteins p62 and LC3B, cells were plated at a density of 5 × 10 5 cells in 60 mm plates in DMEM (without FBS) and incubated for 48 h. After adding 10% FBS, cells were treated with different concentrations of quinoin (5.0, 25, 50,100, and 250 nM) for 24 h. The membranes were incubated with the antibodies anti-p62 and anti-LC3B (1:1000, Cell Signaling Technology, Euroclone, Pero, Italy), anti-Caspase 3 (1:1000, Cell Signaling Technology), and anti-Cyclin D1 (1:1000, Cell Signaling Technology).

Techniques: Western Blot, Activation Assay, Concentration Assay, Expressing, Control

Journal: Oncotarget

Article Title: Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas

doi: 10.18632/oncotarget.9649

Figure Lengend Snippet: A. EAC cell lines OE19 and OE33 were treated with increasing concentrations of the autophagy inhibitor chloroquine (CQ) for 48hr followed by immunoblotting of the autophagy markers p62 and LC3B. Total protein was visualized as loading control. Representative blot is shown. B. Quantification of triplicate repetitions of experiments shown in A.

Article Snippet: The anti-p62/SQSTM1 antibody (MBL rabbit polyclonal, #PM045, LabForce, Nunningen, Switzerland) was diluted 1:9000 in tris buffer and incubated at 95°C for 30 min. Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, product number) per manufacturer's instructions.

Techniques: Western Blot, Control

Journal: Oncotarget

Article Title: Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas

doi: 10.18632/oncotarget.9649

Figure Lengend Snippet: EAC tissue exhibiting A. low p62 cytoplasmic staining, B. high p62 cytoplasmic staining C. both high p62 dot-like and diffuse cytoplasmic staining, and D. high p62 nuclear positivity. Representative images were taken on a Zeiss Axioskop microscope at 40X objective magnification and corrected for brightness. Insert in A. and C. is for better visualization of the hardly visible dots.

Article Snippet: The anti-p62/SQSTM1 antibody (MBL rabbit polyclonal, #PM045, LabForce, Nunningen, Switzerland) was diluted 1:9000 in tris buffer and incubated at 95°C for 30 min. Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, product number) per manufacturer's instructions.

Techniques: Staining, Microscopy

Journal: Oncotarget

Article Title: Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas

doi: 10.18632/oncotarget.9649

Figure Lengend Snippet: Summary of staining patterns

Article Snippet: The anti-p62/SQSTM1 antibody (MBL rabbit polyclonal, #PM045, LabForce, Nunningen, Switzerland) was diluted 1:9000 in tris buffer and incubated at 95°C for 30 min. Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, product number) per manufacturer's instructions.

Techniques: Staining

Journal: Oncotarget

Article Title: Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas

doi: 10.18632/oncotarget.9649

Figure Lengend Snippet: p62 dot-like, p62 cytoplasmic and p62 nuclear staining patterns correlated to clinicopathological features respectively

Article Snippet: The anti-p62/SQSTM1 antibody (MBL rabbit polyclonal, #PM045, LabForce, Nunningen, Switzerland) was diluted 1:9000 in tris buffer and incubated at 95°C for 30 min. Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, product number) per manufacturer's instructions.

Techniques: Staining

Journal: Oncotarget

Article Title: Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas

doi: 10.18632/oncotarget.9649

Figure Lengend Snippet: p62 sum score and clinicopathological features

Article Snippet: The anti-p62/SQSTM1 antibody (MBL rabbit polyclonal, #PM045, LabForce, Nunningen, Switzerland) was diluted 1:9000 in tris buffer and incubated at 95°C for 30 min. Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, product number) per manufacturer's instructions.

Techniques:

Journal: Oncotarget

Article Title: Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas

doi: 10.18632/oncotarget.9649

Figure Lengend Snippet: A. Dot-like, B. cytoplasmic or C. nuclear staining classified as either low or high. D. p62 sum score. For each curve the p-value is displayed on the bottom right-hand corner.

Article Snippet: The anti-p62/SQSTM1 antibody (MBL rabbit polyclonal, #PM045, LabForce, Nunningen, Switzerland) was diluted 1:9000 in tris buffer and incubated at 95°C for 30 min. Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, product number) per manufacturer's instructions.

Techniques: Staining

Journal: Oncotarget

Article Title: Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas

doi: 10.18632/oncotarget.9649

Figure Lengend Snippet: A. Kaplan-Meier survival curve analysis using classification strategy based on subcellular location and expression of p62. The cohort was subdivided into three groups based on their p62 cytoplasmic (including dot-like) and nuclear staining patterns: low p62 cytoplasmic/low p62 nuclear (LL), either low p62 cytoplasmic/high p62 nuclear or vice versa (mixed), high p62 cytoplasmic/high p62 nuclear (HH). B. Kaplan-Meier survival curve analysis using classification strategy based on LC3B dot-like expression and total p62 expression. The cohort was subdivided into three groups: low LC3B/low p62 (LL), either low LC3B/high p62 or vice versa (mixed), high LC3B/high p62 (HH). For each curve the p-value is displayed on the bottom right-hand corner.

Article Snippet: The anti-p62/SQSTM1 antibody (MBL rabbit polyclonal, #PM045, LabForce, Nunningen, Switzerland) was diluted 1:9000 in tris buffer and incubated at 95°C for 30 min. Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, product number) per manufacturer's instructions.

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas

doi: 10.18632/oncotarget.9649

Figure Lengend Snippet: p62 cytoplasmic/p62 nuclear classification was shown to be an independent prognostic parameter in a multivariate analysis encompassing pT category, pN category, absence or presence of distant metastasis, lymphatic invasion and perineural invasion as well as grading

Article Snippet: The anti-p62/SQSTM1 antibody (MBL rabbit polyclonal, #PM045, LabForce, Nunningen, Switzerland) was diluted 1:9000 in tris buffer and incubated at 95°C for 30 min. Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, product number) per manufacturer's instructions.

Techniques:

Journal: Oncotarget

Article Title: Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas

doi: 10.18632/oncotarget.9649

Figure Lengend Snippet: p62 cytoplasmic/p62 nuclear classification was shown to be an independent prognostic parameter in a multivariate analysis encompassing UICC and grading

Article Snippet: The anti-p62/SQSTM1 antibody (MBL rabbit polyclonal, #PM045, LabForce, Nunningen, Switzerland) was diluted 1:9000 in tris buffer and incubated at 95°C for 30 min. Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, product number) per manufacturer's instructions.

Techniques:

Journal: Current Research in Food Science

Article Title: Targeting protein aggregation using a cocoa-bean shell extract to reduce α-synuclein toxicity in models of Parkinson's disease

doi: 10.1016/j.crfs.2024.100888

Figure Lengend Snippet: The CBSE reduces α-syn toxicity in neuroblastoma cells. (A–B) Western blot analysis using anti-α-syn, anti-phospho-T172-AMPK, anti-AMPKα, anti-vinculin antibodies (A) and anti-p62/SQSTM1 anti-phospho-Ser555-ULK1, anti-ULK1 and anti-vinculin antibodies (B) on protein extracts from SH-SY5Y pTet-SNCA-FLAG cells untreated, treated with doxycycline or treated with doxycycline and 150 μg/mL CBSE for 48 and 72 h. (C) Representative immunofluorescence (60x) images of SH-SY5Y cells treated with doxycycline (Doxy) alone and in combination with 150 μg/ml CBSE (Doxy + Cocoa) for 48 h, immunolabeled with anti α-syn antibody (C), A11 anti-oligomer antibody (D), and Proteostat R dye (E). Nuclei were stained by DAPI (Blue). Histograms represent mean ± standard deviation of cell fluorescence quantified with the ImageJ software.

Article Snippet: Western blot analysis was performed using anti-α-syn antibody (Merck), anti-phospho-T172-AMPK antibody (Cell Signaling), anti-AMPKα antibody (Cell signalling), anti-p62/SQSTM1 antibody (Merck), anti-phospho-Ser555-ULK1 antibody (Merck), anti-ULK1 antibody (Calbiochem) and anti-vinculin antibody (Sigma).

Techniques: Western Blot, Immunofluorescence, Immunolabeling, Staining, Standard Deviation, Fluorescence, Software