Journal: Biomaterials Research

Article Title: Autophagy-Regulating, Photothermal Polydopamine-Coated, and Photodynamic Zirconium/Porphyrin-Framed Metal–Organic Frameworks for Enhanced Doxorubicin Therapy in Colon Cancer

doi: 10.34133/bmr.0218

Figure Lengend Snippet: Proliferation and migration study of CT-26 colon cancer (A to C) and autophagy induction after exposure to ZrMOFs (D and E): (a) nontreated, (b) ZrMOF, (c) PD/ZrMOF, and (d) DOX-hyd-PD/ZrMOF. (A) Colony-forming assay of CT-26 cells treated with the NPs with different concentrations for 48 h. (B) Time-dependent cell migration images (NP concentration = 2 μg/ml). Scale bar, 200 μm. (C) Migrated cell numbers at 48 h measured by ImageJ (*** P < 0.001, ** P < 0.002, n = 3). (D) Western blot of autophagy-related protein of Beclin-1, Atg7, p62, LC3, p-mTOR, and mTOR. (E) Fold change of LC3B-ll /LC3B-l analyzed from Western blot. (*** P < 0.001, ** P < 0.005, * P < 0.01, n = 3).

Article Snippet: Subsequently, the PVDF membranes were blocked with tris-buffered saline with Tween 20 (TBST) containing 5% skim milk for 1 h and incubated with various primary antibodies against the following: Beclin-1 (Cell Signaling Technology, 3738), Atg7 (Cell Signaling Technology, 2631), LC3B (Cell Signaling Technology, 3868S), p62 (Cell Signaling Technology, 16177), mamalian target of rapamycin (mTOR, Cell signaling Technology, 2972), phospho-mTOR (Cell Signaling Technology, 2971), and β-actin (Cell Signaling Technology, 4970) overnight at 4 °C.

Techniques: Migration, Concentration Assay, Western Blot

Journal: Frontiers in immunology

Article Title: Palmatine treats urticaria by reducing inflammation and increasing autophagy.

doi: 10.3389/fimmu.2023.1268467

Figure Lengend Snippet: FIGURE 6 Immunofluorescence staining (A) and fluorescence intensity (B) of P62. Beclin-1, LC3-I, LC3-II, P62, and GAPDH protein bands in skin samples from the Control, OVA, OVA+LOR and OVA+PAL groups of rats (C). Graphs showing the levels of expression of Beclin-1 (D), P62 (E), LC3-I (F), and LC3-II (G) and LC3-II/LC3-I (H) in the four rat groups. Data are presented as mean ± standard deviation (SD). #P<0.05, ##P<0.01, ###P<0.001 vs. Control group. *P<0.05, ***P<0.001 vs. OVA group. OVA, ovalbumin; LOR, loratadine; PAL, palmatine.

Article Snippet: The membranes were stained with Rexchip Red S dye for 5 min, washed twice with TBST, permeated with TBS, transferred to a sealing solution containing 5% skimmed milk powder in TBST, and sealed by shaking at room temperature for 1 h. The membrane was washed with TBST, sealed, and incubated overnight at 4°C with primary antibody to beclin-1 (1:1000; 66665-1- Ig, Prointech), LC3 A/B (1:1000; 4108, Prointech), P62 (1:5000; 18420- 1-AP, Prointech), LKB1 (1:1000; 3047, Prointech), AMPK (1:1000; 5831, Proteintech), p-AMPK (1:1000), p70S6K1 (1:5000 ratio), pp70S6K1 (1:500; ab32529, Abcam, Cambridge, UK), Atg5-Atg12 complex (1:1000; AAM79 Bio-Rad, CA, USA), or GAPDH (1:1000). frontiersin.org The membranes were washed three times with TBST, followed by incubation at room temperature for 1 h with goat anti-rat IgG H&L (HRP; 1:10,000, ab150157, Abcam) or goat anti-rabbit IgGH&L (HRP; 1:10,000, ab6721, Abcam).

Techniques: Staining, Control, Expressing, Standard Deviation

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A – D Protein expression of LC3-I/II, p62, Parkin, PINK1 in neuronal PC12 cells detected by western blot. GAPDH was used as a loading control. E Immunofluorescence images represent LC3 dots (green) and nuclei (blue) in neuronal PC12 cells. Scale bar: 10 µm. F , G Quantification of the number of LC3 dots per cell and the percentage of cells with LC3 dots. Data are presented as mean ± SEM. **** P < 0.0001, vs. control group. H Electron microscopy showed autophagy induced by EtOH (5%, vol/vol, 1 and 3 h) in neuronal PC12 cells. The arrows point to the autophagosome (double membrane) and autolysosome (single membrane). Scale bar: 1 µm. I , J Quantification of the number of LC3 dots per cell. Data are presented as mean ± SEM. *** P < 0.001 , **** P < 0.0001, vs. control group. #### P < 0.000, vs. EtOH 1 h group. ^^ P < 0.01, ^^^^ P < 0.0001, vs. EtOH 3 h group. K – N Quantification results of mean neurite length per cell, the percentage of cell with neurite, the percentage of cells with fragmented mitochondria, and TMRM mean fluorescence intensity, which show that the autophagy inhibitor 3-MA had no inhibitory effect on the cell recovery from EtOH induced damage. EtOH ethanol.

Article Snippet: After blocking, membranes were incubated overnight at 4°C with primary antibodies against LC3B (NB100-2220, Novus Biologicals, USA), p62/SQSTM1 (NBP1-48320, Novus Biologicals, USA), PINK1 (BC100-494, Novus Biologicals, USA), Parkin (sc-32282, Santa Cruz, USA), Drp1 (8570 S, Cell Signaling Technology, USA), p-Drp1 (Ser616) (PA5-106169, Invitrogen TM , USA), p-Drp1 (Ser637) (PA5-101038, Invitrogen TM , USA), OPA1 (67589S, Cell Signaling Technology, USA), Mitofusion-1 (14739S, Cell Signaling Technology, USA), Mitofusion-2 (9482S, Cell Signaling Technology, USA), PGC-1α (NB100-60955, Novus Biologicals, USA), AMPK-1α (2532S, Cell Signaling Technology, USA), p-AMPK-1α (2531S, Cell Signaling Technology, USA), SIRT1 (9475S, Cell Signaling Technology, USA), and GAPDH (10R-G109a, Fitzgerald Industries, UK).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Electron Microscopy, Membrane, Fluorescence, Cell Recovery

Journal: Journal of cellular physiology

Article Title: Endoplasmic reticulum stress and unfolded protein response profile in quadriceps of sarcopenic patients with respiratory diseases.

doi: 10.1002/jcp.27789

Figure Lengend Snippet: FIGURE 5 (a) Mean values and 95% confidence intervals of expression levels of the following markers: Atrogin‐1 and MuRF‐1 expressed as relative messenger RNA levels in the vastus lateralis muscle of the study groups: Healthy controls, LC‐cachexia, and COPD patients. (b) Mean values and 95% confidence intervals of LC3B protein content in the vastus lateralis muscle as measured by optical densities in arbitrary units (OD, a.u.). (c) Mean values and 95% confidence intervals of p62 protein content in the vastus lateralis muscle as measured by optical densities in arbitrary units (OD, a.u.). (d) Mean values and 95% confidence intervals of MDA‐protein adducts in the vastus lateralis muscle as measured by optical densities in arbitrary units (OD, a.u.). COPD: chronic obstructive pulmonary disease; LC: lung cancer; LC3B: microtubule‐associated protein 1 light chain 3; MDA: malondialdehyde; MuRF‐1: muscle ring finger protein‐1; p62: nucleoporin p62. *p ≤0.05, **p ≤0.01, and ***p ≤0.001 between either the LC‐cachexia patients or any of COPD patient groups (nonsarcopenic and sarcopenic patients) and the healthy controls

Article Snippet: The following primary antibodies were used: Anti‐binding immunoglobulin protein (BIP; 1:1,000, ab37073) antibody (Abcam, Cambridge, UK), anti‐protein disulfide isomerase‐3 (PDIA3; 1:1,000, ADI‐SPA‐725‐F) antibody (Enzo Life Science, Farmingdale, NY), anti‐phosphatidylinositol 3‐kinase (PI3K; 1:1,000, 3358S) antibody (Cell Signaling, Boston, MA), anti‐ATF6 (1:100, ab37149), anti‐protein kinase R (PKR)‐like endoplasmic reticulum kinase (PERK; 1:1,000, ab79483), anti‐phospho‐PERK (1:1,000, ab192591), anti‐eukaryotic translation initiation factor 2α (eiF2a; 1:500, ab181467), anti‐phospho‐eiF2a (1:1,000, ab32157), anti‐C/ EBP‐homologous protein (CHOP; 1:2,000, ab11419), anti‐IRE1 (1:1,000, ab37073), antitumor necrosis factor receptor‐associated factor 2 (TRAF2; 1:1,000, ab37118), anti‐X‐box binding protein 1 (XBP1; 1:1,000, ab37152) antibodies from Abcam, anti‐microtubule‐associated protein 1 light chain 3 (LC3; 1:1,000, #2775S) antibody (Cell Signaling), anti‐nucleoporin p62 (p62; 1:1,000, P0067) antibody (Sigma‐Aldrich, St. Louis, MO), anti‐ apoptosis signal‐regulating kinase 1 (ASK1; 1:500, sc‐5294) antibody from Santa Cruz Biotechnology (Santa Cruz, CA), anti‐malondialdehyde‐ protein adducts (MDA; 1:4,000, MD20A‐G1b) antibody (Academy Bio‐Medical Company, Houston, TX), and anti‐glyceraldehyde‐3‐ phosphate dehydrogenase (GAPDH; 1:2,000, sc‐25778) antibody (Santa Cruz Biotechnology).

Techniques: Expressing

Journal: bioRxiv

Article Title: Reduction of Spermine Synthase Suppresses Tau Accumulation Through Autophagy Modulation in Tauopathy

doi: 10.1101/2023.03.17.533015

Figure Lengend Snippet: (A) Western-blot of Tau, EGFP, autophagy marker LC3, cargo recruiter p62 and SMS in SH-SY5Y cells with Tau/EGFP plasmids and Control/SMS siRNA transfection. The image is a representative of four separate experiments. (B) Quantification of the protein levels of Tau (5A6), EGFP, LC3-I (cytoplasmic), LC3-II (autophagosome-associated), p62 and SMS in (A). All the protein levels were normalized with the β-Actin level. All the values were further normalized by that of the control cells. n = 4; Student’s t test (Tau or EGFP) or two-way ANOVA multiple comparisons (others). (C) p62 staining and Alexa 647-conjugated Tau K18 fibrils in SVG p12 cells with Control/SMS siRNA transfection. The images are representatives of five fields. (D) Quantification of the size (area) and intensity of the Alexa 647 conjugated Tau K18 fibrils in (C). n = 5; Student’s t test. Data represent mean ± SEM.

Article Snippet: The following commercially available antibodies were used: anti-GABARAP for Drosophila Atg8a (PM037, MBL), anti-Ref(2)P (ab178440, Abcam), anti-Tau 5A6 (5A6, DSHB), anti-Phospho-Tau AT8 (MN1020, Thermo), anti-cleaved caspase 3 (9661, Cell Signaling), anti-LC3B (L7543, Sigma), anti-p62 (NBP1-48320, Novus Biologicals), anti-GFP (G5144, Invitrogen), anti-Actin (A1978, Sigma), Cy5-conjugated anti-HRP (123175021, Jackson ImmunoLab), and secondary antibodies conjugated to Alexa 488/568/647 (Thermo Fisher Scientific), or near infrared (IR) dye 700/800 (Rockland).

Techniques: Western Blot, Marker, Control, Transfection, Staining

Journal: Journal of Clinical Investigation

Article Title: PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis

doi: 10.1172/jci74942

Figure Lengend Snippet: Figure 3. Defective autophagy in AECIIs from IPF lungs. (A) Representative immunostaining of lung sections from donor and IPF patients using anti-LC3 (red; autophagosomal marker) and anti–ATP synthase (green; mitochondrial marker) antibodies. Yellow puncta denote colocalization. Scale bars: 10 μm. (B) x-z coordinate image of z stack of merged LC3 and ATP synthase image of the IPF lung section in A. Partial colocalization was seen for the mitochondrial and autophagosomal markers (arrow). (C) Western blot analyses of p62 and LC3I/LC3II in isolated AECIIs from donor age-matched control and IPF lungs. Each lane represents an individual AECII preparation. Blots were stripped and reblotted using an anti–β-actin antibody as loading control. Results are also quantified below. Data represent mean ± SEM. *P < 0.05, unpaired, 2-tailed Student’s t test. (D) Representative immunostaining of donor and IPF patient lung sections using anti–SP-C (green) and anti-p62 (red). Yellow indicates colocaliza- tion of the markers. Scale bars: 10 μm.

Article Snippet: The antibodies used were as follows: MFN1, TOM20, β-actin, DRP1, p-JNK, and BAX (all Santa Cruz); α-tubulin, p-DRP1, JNK, AKT, and p-AKT (all Cell Signaling); OPA1, p62, and MFN2 (all Abcam); PINK1 (Novus); LC3 (MBL); PTEN (Millipore).

Techniques: Immunostaining, Marker, Western Blot, Isolation, Control

Journal: Journal of Clinical Investigation

Article Title: PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis

doi: 10.1172/jci74942

Figure Lengend Snippet: Figure 5. Stimulation of ER stress deteriorates mitochondrial function and impairs mitophagy in lung epithelial cells. (A) A549 cells were treated with or without TM (1 μg/ml for 24 hours), and mitochon- drial mass was determined by MitoTracker Green. Induction of autophagy by serum starvation reduced mitochondrial mass in TM-treated cells. The autoph- agy inhibitor bafilomycin A1 increased mitochondrial mass in untreated and TM-treated cells. (B) TM induced dose-dependent depolarization of mito- chondria in A549 cells (assessed by JC-1 dye staining). Depolarization was increased in the presence of bafilomycin A1, but was not affected by starvation conditions. (C) Increased doses of TM induce apop- tosis of A549 cells (assessed by annexin V staining). (D) Representative Western blot analyses showing increased levels of the mitochondrial marker TOM20 and autophagy markers p62 and LC3I/LC3II in lung lysates from aging and young mice after vehicle and TM treatment (2 μg/mouse). The β-actin blot was obtained from parallel samples run on a separate gel from the TOM20 and p62 blots. (E) Density analyses of Western blots in D. Data represent mean ± SEM (A–C and E). *P < 0.05, **P < 0.01, 1- (A–C) or 2-way (E) ANOVA with post-hoc Bonferroni.

Article Snippet: The antibodies used were as follows: MFN1, TOM20, β-actin, DRP1, p-JNK, and BAX (all Santa Cruz); α-tubulin, p-DRP1, JNK, AKT, and p-AKT (all Cell Signaling); OPA1, p62, and MFN2 (all Abcam); PINK1 (Novus); LC3 (MBL); PTEN (Millipore).

Techniques: Staining, Western Blot, Marker

Journal: Journal of Clinical Investigation

Article Title: PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis

doi: 10.1172/jci74942

Figure Lengend Snippet: Figure 10. Mitochondrial dysfunction and increased cell apoptosis in PINK1-deficient mice. (A) Complex I and complex IV activity, both basal and after MHV68 infection, was reduced in Pink1–/– versus Pink1+/+ lung mitochondria. CS, citrate synthase. (B) Mitochondrial mass (assessed by mtDNA/gDNA ratio) in lungs of infected Pink1+/+, Pink1+/–, and Pink1–/– mice. (C) Representative in situ TUNEL assay in lung sections at day 15 after MHV68 infection. Note the increase in positive signal (brown) in PINK1-deficient lungs. Scale bars: 50 μm. (D) Semiquantitative analyses showed significantly higher TUNEL-positive signal in PINK1-deficient versus control mice. (E and F) Immunoblot analyses in whole lung lysates from naive (E) and MHV68-infected (F) Pink1+/+, Pink1+/–, and Pink1–/– mice for BAX, OPA1, and the autophagic markers LC3I/LC3II and p62. Blots were stripped and reblotted with β-actin for loading normalization. Each lane represents an individual mouse. (G) Density analyses of LC3 and p62. Data represent mean ± SEM (A, B, D, and G). #P < 0.05 vs. Pink1+/+; *P < 0.05; 1- (B and D) or 2-way (A and G) ANOVA with post-hoc Bonferroni.

Article Snippet: The antibodies used were as follows: MFN1, TOM20, β-actin, DRP1, p-JNK, and BAX (all Santa Cruz); α-tubulin, p-DRP1, JNK, AKT, and p-AKT (all Cell Signaling); OPA1, p62, and MFN2 (all Abcam); PINK1 (Novus); LC3 (MBL); PTEN (Millipore).

Techniques: Activity Assay, Infection, In Situ, TUNEL Assay, Control, Western Blot