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: Journal of Oncology

Article Title: Exosomal CTCF Confers Cisplatin Resistance in Osteosarcoma by Promoting Autophagy via the IGF2-AS/miR-579-3p/MSH6 Axis

doi: 10.1155/2022/9390611

Figure Lengend Snippet: Exosomal CTCF-mediated IGF2-AS activates autophagy to enhance CDDP resistance of OS cells. (a) Detection of the silencing efficiency of lentiviral sh-IGF2-AS in MG63 cells by RT-qPCR. (b) Determination of the expression of CTCF and IGF2-AS in the parental cells by RT-qPCR and Western blot analysis. (c) CCK-8 applied to detect the IC50 value of OS cells to CDDP. (d) Flow cytometry adopted for detection of the apoptosis in OS cells. (e) TEM data of autophagy in MG63 and MG63/CDDP cells after the intervention of CDDP; the red arrow was the autophagosome. (f) Western blot analysis to measure the expression of autophagy-related genes p62, Beclin1, and LC3 in MG63 and MG63/CDDP cells after CDDP intervention. (g) Autophagy in the cells observed by TEM after the intervention of the autophagy agonist rapamycin. (h) The expression of autophagy-related genes p62, Beclin1, and LC3 in cells determined by Western blot analysis after the intervention of the autophagy agonist rapamycin. (i) CCK-8 used to examine the IC50 value of OS cells to CDDP after rapamycin intervention. (j) Flow cytometry to detect the apoptosis of OS cells after rapamycin intervention. ∗ p < 0.05 vs. sh-NC/control/MG63, # p < 0.05 vs. Exo-CTCF+sh-NC, and & p < 0.05 vs. Exo-CTCF+sh-IGF2-AS. Cell experiment was repeated three times.

Article Snippet: After separation, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (1620177, Bio-Rad) and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. The membrane was probed with primary antibodies to CTCF (ab128873, 1 : 5000, rabbit), MSH6 (ab208940, 1 : 1000, rabbit), p62 (ab109012, 1 : 10000, rabbit), Beclin1 (Ab210498, 1 : 1000, rabbit), and LC3 (ab192890, 1 : 2000, rabbit) overnight at 4°C.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Flow Cytometry, Control

Journal: Journal of Oncology

Article Title: Exosomal CTCF Confers Cisplatin Resistance in Osteosarcoma by Promoting Autophagy via the IGF2-AS/miR-579-3p/MSH6 Axis

doi: 10.1155/2022/9390611

Figure Lengend Snippet: IGF2-AS/miR-579-3p/MSH6 axis activates the autophagy signaling pathway to enhance CDDP resistance of OS cells. (a) RT-qPCR and Western blot analysis for determination of the silencing effect of lentiviral sh-MSH6. (b) Expressions of IGF2-AS, miR-579-3p, and MSH6 in OS cells determined by RT-qPCR and Western blot analysis. (c) The autophagy in the OS cells observed under TEM. (d) Measurement of expressions of autophagy-related genes p62, Beclin1, and LC3 in OS cells by Western blot analysis. (e) The CCK-8 assay to detect the IC50 value of CDDP in OS cells. (f) Flow cytometry analysis to examine the apoptosis of OS cells. ∗ p < 0.05 vs. sh-NC/oe-NC+sh-NC, # p < 0.05 vs. oe-IGF2-AS+sh-NC. Cell experiment was repeated three times.

Article Snippet: After separation, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (1620177, Bio-Rad) and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. The membrane was probed with primary antibodies to CTCF (ab128873, 1 : 5000, rabbit), MSH6 (ab208940, 1 : 1000, rabbit), p62 (ab109012, 1 : 10000, rabbit), Beclin1 (Ab210498, 1 : 1000, rabbit), and LC3 (ab192890, 1 : 2000, rabbit) overnight at 4°C.

Techniques: Quantitative RT-PCR, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Journal of Oncology

Article Title: Exosomal CTCF Confers Cisplatin Resistance in Osteosarcoma by Promoting Autophagy via the IGF2-AS/miR-579-3p/MSH6 Axis

doi: 10.1155/2022/9390611

Figure Lengend Snippet: Exosomal CTCF activated the autophagy signaling pathway to facilitate CDDP resistance of OS cells in an IGF2-AS/miR-579-3p/MSH6-dependent manner. (a) RT-qPCR and Western blot analysis for determination of expression of CTCF, IGF2-AS, miR-579-3p, and MSH6 in MG63 cells. (b) Observation of autophagy numbers in MG63 cells under a TEM. (c) Expression of autophagy-related genes p62, Beclin1, and LC3 in MG63 cells measured by Western blot analysis. (d) The CCK-8 assay to evaluate the sensitivity of MG63 cells to CDDP. (e) Apoptosis of MG63 cells assessed by flow cytometry. ∗ p < 0.05 vs. sh-NC, # p < 0.05 vs. Exo-CTCF+sh-NC. Cell experiment was repeated three times.

Article Snippet: After separation, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (1620177, Bio-Rad) and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. The membrane was probed with primary antibodies to CTCF (ab128873, 1 : 5000, rabbit), MSH6 (ab208940, 1 : 1000, rabbit), p62 (ab109012, 1 : 10000, rabbit), Beclin1 (Ab210498, 1 : 1000, rabbit), and LC3 (ab192890, 1 : 2000, rabbit) overnight at 4°C.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Downregulation of miR-335-5P in Amyotrophic Lateral Sclerosis Can Contribute to Neuronal Mitochondrial Dysfunction and Apoptosis

doi: 10.1038/s41598-020-61246-1

Figure Lengend Snippet: Mitochondria co-localize with p62 after miR-335 inhibitor transfection. ( A ) Co-localization of mitochondria (red) with p62 autophagic marker (green), 72 h after transfection with miR-335 inhibitor. ( B ) Representative WB of p62 in cells after 24, 48 or 72 hours after transfection. Bands corresponding to p62 and β-actin were cropped from the same gel. ( C ) WB quantification showed a decrease in p62 expression at 48 h, and an increase at 72 h after transfection, indicating potential impairment of autophagy in transfected cells. The graphic representation was created using GraphPad Prism, version 5.03, ( https://www.graphpad.com/scientific-software/prism/ ).

Article Snippet: The blots were incubated for 1 hour with the primary antibodies rabbit anti-caspase-3 (1/250 dilution) (Genetex, CA, USA, Cat#GTX110543, RRID:AB_10722709), mouse monoclonal anti-caspase-7 (1/250 dilution) (Thermo Fisher Scientific, Cat#MA1-91896, RRID:AB_2068163) and rabbit anti-p62 (1/250 dilution)(Enzo LifeSciences, Cat# BML-PW9860, RRID:AB_2196009).

Techniques: Transfection, Marker, Expressing, Software

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: Cells

Article Title: Around-the-Clock Noise Induces AD-like Neuropathology by Disrupting Autophagy Flux Homeostasis

doi: 10.3390/cells11172742

Figure Lengend Snippet: Around-the-clock noise (24 h/day) exposure increases AP formation. ( A , E ) Western blot of p62 and LC3B expression in the cortex and hippocampus. ( B , C , F , G ) Quantification data showed that around-the-clock noise (24 h/day) increases the expression of p62 and LC3B in the cortex and hippocampus ( n = 6 per group). ( D , H ) The expression of LC3B at the RNA level in the cortex and hippocampus ( n = 4 per group). ( I , J ) The ultrastructural hippocampus changes were observed with transmission electron microscopy (scale bar: 1μm). * p < 0.05.

Article Snippet: The following primary antibodies were used: mouse anti-β Amyloid (B4) (sc-28365 1:1000, Santa Crutz Biotechnology, Dallas, TX, USA), rabbit anti-Tau (phospho S396) antibody (ab109390, 1:10,000, Abcam, Cambridge, UK), rabbit anti-Tau (phospho S404) antibody (ab109390, 1:10,000, Abcam, Cambridge, UK), mouse GFAP (Ser38) (AP0227 1:1000, Bioworld Technology, Louis Park, MN, USA), rabbit anti-P-AMPK-Thr172 antibody (2535, 1:1000, Cell Signaling Technologies, Danvers, MA, USA), rabbit anti-AMPK antibody (5832, 1:1000, Cell Signaling Technologies, Danvers, MA, USA), rabbit anti-mTOR antibody (P2476, 1:1000, Bioworld Technology, Louis Park, MN, USA), rabbit anti-p-mTOR-(Ser2448) antibody (5536, 1:1000, Cell Signaling Technologies, Danvers, MA, USA), rabbit anti-ULK1 antibody (ab240916, 1:1000, Abcam, Cambridge, UK), rabbit anti-LC3B antibody (ab192890, 1:2000, Abcam, Cambridge, UK), rabbit anti-SQSTM1\P62 antibody (AP6006, 1:1000, Bioworld Technology, Louis Park, MN, USA), rabbit anti-LAMP1 antibody (ab13523, 1:1000, Abcam, Cambridge, UK), rabbit anti-Cathepsin B antibody (31718, 1:1000, Cell Signaling Technologies, Danvers, MA, USA), and mouse anti-GAPDH antibody (MB001, 1:40,000, Bioworld Technology, Louis Park, MN, USA).

Techniques: Western Blot, Expressing, Transmission Assay, Electron Microscopy

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: