Journal: Annals of clinical and translational neurology

Article Title: Clinical heterogeneity within the ALS-FTD spectrum in a family with a homozygous optineurin mutation.

doi: 10.1002/acn3.52075

Figure Lengend Snippet: Figure 4. (A–C): Rt-PCR revealed OPTN-mRNA expression in PBMCs from the brother of the index patient (listed as Pat2, A). On a protein level, optineurin could not be detected in PMBC lysates (B) or lysates from postmortem frontal cortex of the index patient (listed as Pat1) in western blot analysis (C). (D–I) Anti-phospho-TDP-43 immunohistochemistry on spinal cord sections of a sporadic case of ALS (D–F) and of the index patient ALS and the optineurin mutation (G–I). Phospho-TDP43-positive neuronal skein-like and compact cytoplasmic inclusions in motor neurons in the control case (D) are also immunopositive (arrows) for two different anti-optineurin antibodies (E: non-C-terminal; F: C-terminal). In contrast, in the patient with the optineurin mutation, the inclusions (G) do not immunoreact with any of the two anti-optineurin antibodies (H and I). Scale bars: 50 lm: D–F; 100 lm: G–I;

Article Snippet: The Trans-Blot TurboTM Transfer System was used for transfer of proteins onto nitrocellulose membranes at 25 V for 15 min. Membranes with loaded proteins were incubated with EveryBlot Blocking Buffer (Bio-Rad) at room temperature for 30–60 min, followed by incubation with primary antibodies at 4°C for at least 8 h. We used two OPTN polyclonal antibodies (Proteintech, Cat. No. 10837-1-AP, at a dilution of 1:10,000 & Cayman, Item No. 100000, at a dilution of 1:1000), of which one binds to the C-terminus of optineurin (Cayman), and a GAPDH antibody (Cell Signaling, Cat No. 2118, at a dilution of 1:1000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemistry, Mutagenesis, Control

Journal: Chemical Engineering Journal

Article Title: A hydrogel containing Mg2+ with improved osteogenesis, enhanced endochondral ossification, and modulated inflammation for bone-repair applications

doi: 10.1016/j.cej.2024.152762

Figure Lengend Snippet: Fig. 3. Cell viability and osteogenesis of hBMSCs after culturing with OD/CC and OD/CC/AS-Mg2+ hydrogels: (A) Optical density after co-culturing with hydrogels for 1, 3, and 7 d; (B) F-actin levels at day 7; (C) The relative protein levels of COL-1, OCN, OPN, and RUNX2 at day 7 were determined by immunoblotting; (D) ALP activity at day 7; (E) Calcium deposition at day 21; (F)The relative mRNA levels of COL-1, OCN, OPN, and RUNX2 at day 7 were determined by qRT-PCR;(G) Semi- quantitative analysis of Alizarin Red S staining. *p < 0.05, **p < 0.01, ****p＜0.0001, compared to control; #p＜0.05, ##p < 0.01, compared to OD/CC hydrogel. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The RapidStepTM ECL Reagent and polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany), the primary antibodies of COL-I (ab270993), CD31 (ab222783), F-actin (ab205), OCN (ab93876), OPN (ab218237) and RUNX2 (ab76956) (Abcam, Shanghai, China), CD80 (sc-376012) (Santa Cruz Biotechnology Inc., TX, USA), OCN (23418–1-AP) and GAPDH (60004–1-IG) (Proteintech Group, Inc., Wuhan, China), the secondary antibody horseradish peroxidase–labeled poly anti-rabbit IgG (Boster Biological Technology Co. Ltd., Wuhan, China), fluorescein isothiocyanate (FITC)-labeled anti-mouse/anti-rabbit secondary antibodies, and rabbit IgG (Beyotime Biotechnology Co. Ltd., Shanghai, China) were used for immunobloting, immunofluorescent (IF) staining, or immunohistochemical (IHC) staining.

Techniques: Western Blot, Activity Assay, Quantitative RT-PCR, Staining, Control

Journal: Biomedicines

Article Title: Exploratory Analysis of Autophagy–Lysosomal Pathway Proteins in Dermal Fibroblasts as Potential Peripheral Biomarkers for Alzheimer’s Disease: A Pilot Study

doi: 10.3390/biomedicines14010034

Figure Lengend Snippet: Expression profiles of AD- and ALP-associated proteins in AD patient fibroblasts. ( a ) Representative images of Coomassie brilliant blue-stained membranes showing equal protein loading across samples from control and AD patients. ( b ) Representative immunoblot images of α-Tubulin used as a loading control to verify equal protein loading in fibroblasts from control and AD patients. ( c ) Immunoblot analysis of AD- and ALP-associated proteins (APP, BACE1, BAG2, BAG3, Cathepsin D, EEA1, GRP78, LAMP2A, NBR1, OPTN, PSEN2, RAB7, TAX1BP1, TFE3, TOLLIP, and Ubiquitin) in fibroblasts from control ( n = 9) and AD patients ( n = 9). ( d , e ) Quantification of BACE1 ( d ) and TAX1BP1 ( e ) protein levels. Statistical significance was determined using Mann–Whitney U test ( p < 0.05). Violin plots show the distribution; overlaid dots represent individual subject-derived fibroblast lines (biological replicates). The central line indicates the median and the bounds of the box indicate the interquartile range (IQR).

Article Snippet: The following primary antibodies were used: alpha-Tubulin (Abcam, Cambridge, UK; ab18251), APP-CTF (Abcam, Cambridge, UK; ab32136), Beta-site APP cleaving enzyme 1 (BACE1) (ProteinTech Group, Rosemont, IL, USA; 12807-1-AP), BCL2-associated athanogene proteins 2 (BAG2) (Bethyl, Montgomery, TX, USA; A304-751A), BCL2-associated athanogene proteins 3 (BAG3) (ProteinTech Group, Rosemont, IL, USA; 10599-1-AP), Cathepsin D (Calbiochem, San Diego, CA, USA; IM-03), Early endosome antigen 1 (EEA1) (BD Biosciences, San Jose, CA, USA; 610456), Glucose-regulated protein 78 (GRP78) (BD Biosciences, San Jose, CA, USA; 610978), Lysosome-associated membrane protein 2A (LAMP2A) (Abcam, Cambridge, UK; ab18528), Microtubule-associated protein 1 light chain 3 (LC3) (Sigma, St. Louis, MO, USA; L8918), Neighbor of BRCA1 gene 1 (NBR1) (ProteinTech Group, Rosemont, IL, USA; 16004-1-AP), Optineurin (OPTN) (Bethyl, Montgomery, TX, USA; A301-831A), PSEN2 (Bethyl, Montgomery, TX, USA; A304-342A), RAS-Associated Protein RAB7A (RAB7) (Cell Signaling Technologies, Danvers, MA, USA; 9367), Tax1-binding protein 1 (TAX1BP1) (ProteinTech Group, Rosemont, IL, USA; 14424-1-AP), Transcription factor E3 (TFE3) (Bethyl, Montgomery, TX, USA; A302-622), Toll-interacting protein (TOLLIP) (ProteinTech Group, Rosemont, IL, USA; 11315-1-AP), Ubiquitin (ProteinTech Group, Rosemont, IL, USA; 10201-2-AP).

Techniques: Expressing, Staining, Control, Western Blot, Ubiquitin Proteomics, MANN-WHITNEY, Derivative Assay

Journal: Biomedicines

Article Title: Exploratory Analysis of Autophagy–Lysosomal Pathway Proteins in Dermal Fibroblasts as Potential Peripheral Biomarkers for Alzheimer’s Disease: A Pilot Study

doi: 10.3390/biomedicines14010034

Figure Lengend Snippet: Diagnostic performance of fibroblast biomarker models. ( a ) ROC curves assessing the diagnostic performance of individual proteins (BACE1, TAX1BP1, and BAG2) for discriminating AD patients from controls. ( b ) ROC curves showing protein combinations (BAG2 + OPTN and BACE1 + TFE3). ( c ) ROC curves for combinations of APOE4 status with individual protein markers or autophagic flux. Area under curve (AUC), sensitivity, specificity and other statistical parameters are detailed in . Note: Performance estimates may be optimistic due to small sample size and lack of independent validation.

Article Snippet: The following primary antibodies were used: alpha-Tubulin (Abcam, Cambridge, UK; ab18251), APP-CTF (Abcam, Cambridge, UK; ab32136), Beta-site APP cleaving enzyme 1 (BACE1) (ProteinTech Group, Rosemont, IL, USA; 12807-1-AP), BCL2-associated athanogene proteins 2 (BAG2) (Bethyl, Montgomery, TX, USA; A304-751A), BCL2-associated athanogene proteins 3 (BAG3) (ProteinTech Group, Rosemont, IL, USA; 10599-1-AP), Cathepsin D (Calbiochem, San Diego, CA, USA; IM-03), Early endosome antigen 1 (EEA1) (BD Biosciences, San Jose, CA, USA; 610456), Glucose-regulated protein 78 (GRP78) (BD Biosciences, San Jose, CA, USA; 610978), Lysosome-associated membrane protein 2A (LAMP2A) (Abcam, Cambridge, UK; ab18528), Microtubule-associated protein 1 light chain 3 (LC3) (Sigma, St. Louis, MO, USA; L8918), Neighbor of BRCA1 gene 1 (NBR1) (ProteinTech Group, Rosemont, IL, USA; 16004-1-AP), Optineurin (OPTN) (Bethyl, Montgomery, TX, USA; A301-831A), PSEN2 (Bethyl, Montgomery, TX, USA; A304-342A), RAS-Associated Protein RAB7A (RAB7) (Cell Signaling Technologies, Danvers, MA, USA; 9367), Tax1-binding protein 1 (TAX1BP1) (ProteinTech Group, Rosemont, IL, USA; 14424-1-AP), Transcription factor E3 (TFE3) (Bethyl, Montgomery, TX, USA; A302-622), Toll-interacting protein (TOLLIP) (ProteinTech Group, Rosemont, IL, USA; 11315-1-AP), Ubiquitin (ProteinTech Group, Rosemont, IL, USA; 10201-2-AP).

Techniques: Diagnostic Assay, Biomarker Discovery

Journal: BMC Cell Biology

Article Title: Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells

doi: 10.1186/1471-2121-12-11

Figure Lengend Snippet: LPA and ATX/LPC induce OPN expression in SGC7901 cells . (A) Time course of OPN stimulation utilizing ATX (50 ng/ml) + LPC (20 μM) (ATX/LPC2). OPN expression was examined in SGC7901 cells by Western blot and real time-PCR. SGC7901 cells were treated with ATX, LPC, LPA, ATX/LPC1, ATX/LPC2 or DMEM/0.1% BSA. Total protein and RNA was extracted for Western blot (C) and real time -PCR (B), respectively. Figure1 D showed the densitometric ratio of OPN protein/α-tubulin. Statistical analysis was performed using Student's t test. *p < 0.05. The ATX/LPC concentration curve for stimulated motility provides a justification for the concentrations of ATX/LPC chosen throughout the manuscript. LPA or ATX/LPC stimulates OPN production in additional cell lines (eg, SMMC7721 in our study: ATX-LPA axis induces expression of OPN in hepatic cancer cell SMMC7721) as well as in SGC7901 cells.

Article Snippet: ATX (recombinant human ENPP-2/Autotaxin) and OPN antibody were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: BMC Cell Biology

Article Title: Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells

doi: 10.1186/1471-2121-12-11

Figure Lengend Snippet: ATX/LPC-regulated OPN expression is mediated by LPA2 receptor and activation of Akt and MAPK/ERK . SGC7901 cells were exposed to ATX, LPC, LPA, ATX/LPC2 or DMEM/0.1% BSA for 24 hours or 30 min, and then total RNA and protein were extracted for real time-PCR (A-D) and Western blot (E, F), respectively. (E) Immunoblotting was performed to detect the phosphorylation status of ERK and Akt. Total ERK and Akt levels were assessed as control. (F) The effects of the LPA inhibitor, ERK inhibitor and Akt inhibitor on ATX/LPC2-induced OPN expression in SGC7901 cells. SGC7901 cells were treated with Ki16425, PD98059, LY294002 or DMSO (0.1%; a solvent control) for 45 min prior to ATX/LPC2 treatment. 24 hours following stimulations, OPN expression was measured by Western blot analysis. All experiments were performed in triplicate and a representative result is shown here. Figure 2G showed the densitometric ratio of OPN protein/α-tubulin. Statistical analysis was performed using Student's t test. *p < 0.05.

Article Snippet: ATX (recombinant human ENPP-2/Autotaxin) and OPN antibody were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Control, Solvent

Journal: BMC Cell Biology

Article Title: Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells

doi: 10.1186/1471-2121-12-11

Figure Lengend Snippet: Requirement of OPN in migration induced by ATX-LPA axis protects against Taxol-induced apoptosis in SGC7901 cells . (A) Verification of siRNA knockdown of the OPN protein by Western blot showed a significant reduction of OPN protein in clone3 (SGC7901-siRNA-OPN). (B) The effect of OPN on ATX-LPA-induced migration was assayed using a Transwell assay. Either SGC7901, SGC7901-siRNA-vehicle, or SGC7901-siRNA-OPN cells (1 × 10 5 ) were added in the upper chambers of the transwells, incubated in DMEM/0.1% BSA or medium containing ATX, LPC, LPA, ATX/LPC2 and allowed to migrate for 48 hours at 37°C. Migrated cells on the lower chamber were quantified. (D) Flow cytometric analysis of apoptosis: SGC7901, SGC7901-siRNA-vehicle, or SGC7901-siRNA-OPN cells were treated with Taxol (50 nM) in the absence or presence of each group: ATX, LPC, LPA, ATX/LPC2 or DMEM/0.1% BSA for 24 hours. Apoptosis was analyzed by flow cytometry. Data shown in Figure 4C and E represents the mean ± SD from three individual experiments. Data are mean ± SD of triplicate determinations. Statistical analysis was performed using Student's t test. *p < 0.05.

Article Snippet: ATX (recombinant human ENPP-2/Autotaxin) and OPN antibody were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Knockdown, Western Blot, Transwell Assay, Incubation, Flow Cytometry

Journal: The FASEB Journal

Article Title: Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy

doi: 10.1096/fj.201801749rrr

Figure Lengend Snippet: Figure 1. Patients with DN have reduced renal expression of OPTN, and the OPTN expression level correlates negatively with NLRP3 inflammasome activation. A) Immunohistochemical analysis of OPTN expression in RTECs of a representative control patient and a representative patient with type 2 DN. B) OPTN staining scores. Data are expressed as means 6 SD for the control (continued on next page)

Article Snippet: PTECs were transfected with a green fluorescent protein (GFP)-tagged pAdTrack-Vector adenovirus [multiplicity of infection (MOI): 50], a GFP-tagged pAdTrack-OPTN adenovirus (MOI: 50), or a GFP-tagged pAdTrack-OPTN-ubiquitinassociated domains adenovirus (MOI: 50) for 48 h. To silence OPTN expression, murine PTECs were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and control small interferingRNA (siRNA) (sc-36869; SantaCruz Biotechnology) or OPTN siRNA (sc-39055; Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Immunohistochemical staining, Control, Staining

Journal: The FASEB Journal

Article Title: Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy

doi: 10.1096/fj.201801749rrr

Figure Lengend Snippet: Figure 2. HG stimulation of murine RTECs decreases OPTN expression, activates NLRP3 inflammasome, and increases mitochondrial dysfunction. A) Expression of OPTN mRNA in murine RTECs treated with NG for 72 h, high mannitol (HM) for (continued on next page)

Article Snippet: PTECs were transfected with a green fluorescent protein (GFP)-tagged pAdTrack-Vector adenovirus [multiplicity of infection (MOI): 50], a GFP-tagged pAdTrack-OPTN adenovirus (MOI: 50), or a GFP-tagged pAdTrack-OPTN-ubiquitinassociated domains adenovirus (MOI: 50) for 48 h. To silence OPTN expression, murine PTECs were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and control small interferingRNA (siRNA) (sc-36869; SantaCruz Biotechnology) or OPTN siRNA (sc-39055; Santa Cruz Biotechnology).

Techniques: Expressing

Journal: The FASEB Journal

Article Title: Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy

doi: 10.1096/fj.201801749rrr

Figure Lengend Snippet: Figure 3. Overexpression of OPTN inhibits NLRP3 inflammasome activation in HG-treated RTECs. Mouse PTECs were transfected with GFP-tagged pAdTrack-vector adenovirus (50 MOI; control vector) or GFP-tagged pAdTrack-OPTN adenovirus (50 MOI) for 48 h. A) Expression of OPTN was determined by real-time quantitative PCR. B) Representative Western blots of OPTN expression. C) Quantitation of the Western blots in B. D) Representative Western blots of cleaved caspase-1 and cleaved IL-1b in cell supernatants (SN) and of procaspase 1, OPTN, and NLRP3 in whole-cell extracts. E–I) Quantitation of the Western blots in D. J, K) IL-1b (J) and IL-18 (K) levels in cell SN. Values are the mean 6 SD of 3 independent experiments, each with triplicate dishes. *P , 0.05 vs. HG.

Article Snippet: PTECs were transfected with a green fluorescent protein (GFP)-tagged pAdTrack-Vector adenovirus [multiplicity of infection (MOI): 50], a GFP-tagged pAdTrack-OPTN adenovirus (MOI: 50), or a GFP-tagged pAdTrack-OPTN-ubiquitinassociated domains adenovirus (MOI: 50) for 48 h. To silence OPTN expression, murine PTECs were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and control small interferingRNA (siRNA) (sc-36869; SantaCruz Biotechnology) or OPTN siRNA (sc-39055; Santa Cruz Biotechnology).

Techniques: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay

Journal: The FASEB Journal

Article Title: Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy

doi: 10.1096/fj.201801749rrr

Figure Lengend Snippet: Figure 4. Silencing of OPTN enhances NLRP3 inflammasome activation in HG-treated RTECs. Cells were transfected with control siRNA or OPTN siRNA. A) Expression of OPTN mRNA detected by quantitative real-time PCR. B) Representative Western blots of OPTN expression. C) Quantitation of the Western blots in B. D) Representative western blots of cleaved caspase- 1 and cleaved IL-1b in cell supernatants (SN) and procaspase 1 and NLRP3 in whole-cell extracts. E–H) Quantitation of the Western blots in D. I, J) IL-18 (I) and IL-1b (J) levels in cell SN. Values are the mean 6 SD of 3 independent experiments, each with triplicate dishes. *P , 0.05 vs. HG.

Article Snippet: PTECs were transfected with a green fluorescent protein (GFP)-tagged pAdTrack-Vector adenovirus [multiplicity of infection (MOI): 50], a GFP-tagged pAdTrack-OPTN adenovirus (MOI: 50), or a GFP-tagged pAdTrack-OPTN-ubiquitinassociated domains adenovirus (MOI: 50) for 48 h. To silence OPTN expression, murine PTECs were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and control small interferingRNA (siRNA) (sc-36869; SantaCruz Biotechnology) or OPTN siRNA (sc-39055; Santa Cruz Biotechnology).

Techniques: Activation Assay, Transfection, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay

Journal: The FASEB Journal

Article Title: Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy

doi: 10.1096/fj.201801749rrr

Figure Lengend Snippet: Figure 6. OPTN inhibits activation of NLRP3 inflammasome by enhancing mitophagy. A) Representative Western blots of P62 in mitochondrial extracts of OPTN-overexpressing RTECs after HG stimulation for 48 h. B) Quantitation of the Western blots in A. C) Confocal laser scanning microscopy images showing colocalization of translocase of outer mitochondrial membrane 20 (TOMM20) (red) and LC3II (green) as well as location of nuclei (blue). Slanted lines indicate the areas assessed for fluorescence intensity. D) Line scans of fluorescence intensity were measured using ImageJ software, showing colocalization of TOMM20 and LC3II. E) Representative Western blots of cleaved caspase-1 and cleaved IL-1b in cell supernatants (SN) and procaspase1 and NLRP3 in whole- cell extracts. F–I) Quantitation of the Western blots in E. J, K) IL-1b (J) and IL-18 (K) levels in cell SN. Values are the mean 6 SD of 3 independent experiments, each with triplicate dishes. *P , 0.05 vs. HG, &P , 0.05 vs. HG+OPTN-overexpression.

Article Snippet: PTECs were transfected with a green fluorescent protein (GFP)-tagged pAdTrack-Vector adenovirus [multiplicity of infection (MOI): 50], a GFP-tagged pAdTrack-OPTN adenovirus (MOI: 50), or a GFP-tagged pAdTrack-OPTN-ubiquitinassociated domains adenovirus (MOI: 50) for 48 h. To silence OPTN expression, murine PTECs were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and control small interferingRNA (siRNA) (sc-36869; SantaCruz Biotechnology) or OPTN siRNA (sc-39055; Santa Cruz Biotechnology).

Techniques: Activation Assay, Western Blot, Quantitation Assay, Confocal Laser Scanning Microscopy, Membrane, Software, Over Expression

Journal: The FASEB Journal

Article Title: Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy

doi: 10.1096/fj.201801749rrr

Figure Lengend Snippet: Figure 8. Down-regulation of OPTN induces mtROS production and activates NLRP3 inflammasome. A) Fluorescence microscopy of dihydrorhodamine 123 (DHR123) (green, ROS) and DAPI (blue, nuclei) in cells transfected with control siRNA or OPTN-siRNA, treated with NG or HG with or without MitoTempo. B) Quantitation of DHR123 fluorescence. C) Representative Western blots of cleaved caspase-1 and cleaved IL-1b in cell supernatants (SN) and procaspase1 and NLRP3 in whole-cell extracts of RTECs with or without HG and with or without MitoTempo. D–G) Quantitation of the Western blots in C. H, I) IL-1b (H) and IL-18 (I) levels in cell SN. Values are the mean 6 SD of 3 independent experiments, each with triplicate dishes. *P , 0.05 vs. HG, &P , 0.05 vs. HG+OPTN siRNA.

Article Snippet: PTECs were transfected with a green fluorescent protein (GFP)-tagged pAdTrack-Vector adenovirus [multiplicity of infection (MOI): 50], a GFP-tagged pAdTrack-OPTN adenovirus (MOI: 50), or a GFP-tagged pAdTrack-OPTN-ubiquitinassociated domains adenovirus (MOI: 50) for 48 h. To silence OPTN expression, murine PTECs were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and control small interferingRNA (siRNA) (sc-36869; SantaCruz Biotechnology) or OPTN siRNA (sc-39055; Santa Cruz Biotechnology).

Techniques: Fluorescence, Microscopy, Transfection, Control, Quantitation Assay, Western Blot

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: HCT116 and HeLa cells stably expressing po-mKeima were transfected with a plasmid encoding GFP or OPTN-GFP and cultured as described in the Materials and Methods section. After two days, the cells were harvested and processed for FACS analysis. To measure the percentage of cells undergoing pexophagy, single-cell GFP-positive populations were gated for a decrease in fluorescence intensity in the neutral channel. a , Representative flow cytometry plots of each group (n = 3). The different colors represent the cell density at a given position. b , Quantification of the percentage of cells in the gated area. The data are shown as the mean ± SD and represent the values of 3 independent biological replicates. All conditions were statistically compared to the GFP condition (ns, non-significant; **, p < 0.01).

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Cell Culture, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: a , Distribution patterns of OPTN-GFP and mCherry-PTS1 in four distinct cell lines, either co-expressing (right panels) or not (left panels) a high-affinity plasma membrane-targeted anti-GFP nanobody (GFP Nb -PM). Before analysis, the cells were incubated for 2 h with 200 nM BafA1. Representative images are shown. The white arrowheads indicate colocalization of OPTN-GFP and mCherry-PTS1. The yellow arrowheads indicate positions where peroxisomes are associated with the plasma membrane. b , Enlargements of the boxed areas in panel a. c , Distribution patterns of endogenous PEX14 and OPTN in HEK-293 and HCT116 cells. d , High-resolution imaging and volume rendering of two mCherry-PTS1/OPTN-GFP aggregates in HEK-293 cells. Scale bars: a-c , 10 μm; d , 500 nm.

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Expressing, Clinical Proteomics, Membrane, Incubation, Imaging

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: a , Schematic representation of the distinct domains and major interactions of the miniTurbo-OPTN-GFP fusion protein. ATG9A, autophagy-related protein 9A; CC, coiled-coil; CYLD, cylindromatosis; HLH, helix-loop-helix; HTT, huntingtin; LZ, leucine zipper domain; LC3/GABARAP, microtubule-associated protein 1A/1B-light chain 3/γ-aminobutyric acid receptor-associated protein; LIR, LC3-interacting region; Rab, Ras-related protein; TBC1D17, TBC1 domain family member 17; TBK1, TANK (TRAF-associated NF-kB activator) binding kinase 1; UBD, ubiquitin-binding domain; ZF, zinc finger. b , Volcano plot displaying the Log 2 -fold changes (FC) in protein abundance alongside the corresponding Log 10 -fold adjusted p-values (pAdj) for 2856 proteins obtained through streptavidin-affinity purification and identification/quantification by LC–MS/MS. The analysis was performed on miniTurbo-OPTN-GFP-expressing HEK-293 cells (n = 4 biological replicates), assayed under conditions with or without 50 µM biotin. Green, blue, and red dots represent miniTurbo-OPTN-GFP, known OPTN interaction partners, and bona fide peroxisomal proteins that could be identified in the affinity-purified fractions. Vertical and horizontal dotted lines denote the FC (2x) and pAdj (0.05) cut-offs, respectively.

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Binding Assay, Ubiquitin Proteomics, Quantitative Proteomics, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: a , Immunoblot analysis of input and GFP-Trap pull-down fractions from Flp-In T-REx 293 cells expressing the specified proteins. Representative images are shown. Specific protein bands and their degradation products are marked by arrows and arrowheads, respectively. Molecular mass markers (in kDa) are indicated on the left. b , Densitometry quantifications of the relative ratios (RR) of PEX14/GFP and PEX14/OPTN-GFP retained on the GFP-Trap affinity matrix. The total signal intensities of the GFP proteins and PEX14 were both standardized to 100%. The bars represent the mean of 2 biological replicates. The densitometry quantifications of the relative normalized amounts (R-NA) of the proteins under study in the soluble extract input fractions are shown in the . T, transient transfection; F, Flp-In integration; L, lentiviral transduction.

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Western Blot, Expressing, Transfection, Transduction

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: Total cell lysates from lentiviral (L-) transduced Flp-In T-REx 293 cells, which constitutively express either GFP or OPTN-GFP, were subjected to SDS-PAGE followed by immunoblotting using antibodies targeting OPTN and GAPDH. a , A representative image is shown, with specific protein bands indicated by arrows. Note that the 66 kDa band in the OPTN-GFP lane may represent either a degradation product of OPTN-GFP or endogenous OPTN stabilized by overexpression of OPTN-GFP. Molecular mass markers (in kDa) are indicated on the left. b , Densitometry quantifications of the relative normalized amounts (R-NA) of OPTN. The expression levels were normalized to GAPDH, with the amount of OPTN detected in the GFP cells serving as the reference. The amount in the L-OPTN-GFP cells represents the sum of the 66 and 95 kDa bands. The bars represent the mean of 4 biological replicates.

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: SDS Page, Western Blot, Over Expression, Expressing

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: Flp-In T-REx 293 cells stably expressing po-mKeima were transfected with a plasmid encoding the indicated GFP-fusion protein and cultured in regular DMEM medium. After two days, the cells were harvested and processed for GFP-Trap and FACS analyses. To measure the percentage of cells undergoing pexophagy, single-cell GFP-positive populations were gated for a decrease in fluorescence intensity in the neutral channel. a , Samples of protein extracts (input) and the GFP Trap were processed for SDS-PAGE followed by immunoblotting using antibodies to GFP, PEX14, and GAPDH. Representative blots are shown. Specific protein bands are marked by arrows. Molecular mass markers (in kDa) are indicated on the left. b , Densitometry quantifications of the relative ratios (RR) of PEX14/GFP and PEX14/OPTN-GFP fusion proteins retained on the GFP-Trap affinity matrix. The total signal intensities of the GFP proteins and PEX14 were both standardized to 100%. The bars represent the mean of 3 biological replicates. The densitometry quantifications of the relative normalized amounts (R-NA) of the proteins under study in the soluble extract input fractions are shown in the . c , Examples of po-mKeima-expressing cells displaying no, weak, moderate, or excessively high levels of pexophagy. The visuals depict overlay images of po-mKeima excited at 440 nm (false color: green) or 586 nm (false color: red). Yellowish (=low 586/440 excitation peak ratio) and reddish (=high 586/440 excitation peak ratio) dots represent peroxisomes and autolysosomes, respectively . Scale bar, 10 µm. d , Representative flow cytometry plots of each group (n = 3). The different colors represent the cell density at a given position. e , Quantification of the percentage of cells in the gated area. The data are shown as the mean ± SD and represent the values of 3 independent biological replicates. All conditions were statistically compared to the OPTN 1-577 -GFP condition (ns, non-significant; *, p < 0.05; **, p < 0.01; ****, p < 0.0001).

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Cell Culture, Fluorescence, SDS Page, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: Flp-In T-REx 293 cells stably expressing po-mKeima were transfected with a plasmid encoding the indicated GFP-fusion protein and cultured in regular DMEM medium. After two days, the cells were harvested and processed for GFP-Trap and FACS analyses. To measure the percentage of cells undergoing pexophagy, single-cell GFP-positive populations were gated for a decrease in fluorescence intensity in the neutral channel. a , c Samples of protein extracts (input) and the GFP-Trap were processed for SDS-PAGE followed by immunoblotting using antibodies to GFP, PEX14, and GAPDH. Representative blots are shown. Specific protein bands are marked by arrows. Molecular mass markers (in kDa) are indicated on the left. b , d Densitometry quantifications of the relative ratios (RR) of PEX14/GFP and PEX14/OPTN-GFP fusion proteins retained on the GFP-Trap affinity matrix. The total signal intensities of the GFP proteins and PEX14 were both standardized to 100%. The bars represent the mean of 3 biological replicates. The densitometry quantifications of the relative normalized amounts (R-NA) of the proteins under study in the soluble extract input fractions are shown in the . e , Representative flow cytometry plots of each group (n = 3). The different colors represent the cell density at a given position. f , Quantification of the percentage of cells in the gated area. The data are shown as the mean ± SD and represent the values of 3 independent biological replicates. All conditions were statistically compared to the OPTN 1-577 -GFP condition (ns, non-significant; *, p < 0.05; **, p < 0.01; ****, p < 0.0001).

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Cell Culture, Fluorescence, SDS Page, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: Flp-In T-REx 293 cells stably expressing po-mKeima were transfected with either a control (CT) or OPTN DsiRNA and seeded in 6-well plates. On day 2, the cells were transfected or not with a plasmid encoding OPTN 217-577 -GFP. On day 3, the cells were transfected again with either siRNA CT or siRNA OPTN. On day 4, the cells were harvested and processed for immunoblotting with antisera raised against the indicated proteins. a , Representative blots showing the effectiveness of the OPTN DsiRNA. Specific protein bands are marked by arrows. b , Densitometry quantifications of the relative normalized amounts (R-NA) of the OPTN levels shown in panel a (n = 3). c , Comparison of the migration behavior of endogenous OPTN (CT DsiRNA condition) and OPTN 217-577 -GFP (OPTN DsiRNA / OPTN 217-577 -GFP condition). Molecular mass markers (in kDa) are indicated on the left.

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Stable Transfection, Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Comparison, Migration

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: Flp-In T-REx 293 cells, stably expressing po-mKeima, were transfected with either a control (CT) or OPTN siRNA and seeded in 6-well plates. On day 2, the cells were transfected with either a GFP- or OPTN 217-577 -GFP-encoding plasmid. On day 3, the cells were transfected again with either siRNA CT or siRNA OPTN. On day 4, the cells were harvested and processed for FACS analysis. To measure the percentage of cells undergoing pexophagy, single-cell GFP-positive populations were gated for a decrease in fluorescence intensity in the neutral channel. a , Representative flow cytometry plots of each group (n = 3). The different colors represent the cell density at a given position. b , Quantification of the percentage of cells in the gated area. The data are shown as the mean ± SD and represent the values of 3 independent biological replicates. The impact of OPTN downregulation and OPTN 217-577 -GFP overexpression were statistically compared (ns, non-significant; *, p < 0.05; **, p < 0.01;****, p < 0.0001).

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Stable Transfection, Expressing, Transfection, Control, Plasmid Preparation, Fluorescence, Flow Cytometry, Over Expression

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: Flp-In T-REx 293 cells stably expressing po-mKeima were (co-)transfected with (a) plasmid(s) encoding the indicated protein(s) and cultured in regular DMEM medium. After two days, the cells were harvested and processed for GFP-Trap and FACS analysis. To measure the percentage of cells undergoing pexophagy, single-cell GFP-positive populations were gated for a decrease in fluorescence intensity in the neutral channel. a , Samples of protein extracts (input) and the GFP-Trap were processed for SDS-PAGE followed by immunoblotting using antibodies to GFP, OPTN, and GAPDH. Representative blots are shown. Specific protein bands are marked by arrows. Molecular mass markers (in kDa) are indicated on the left. b , Densitometry quantifications of the relative ratios (RR) of OPTN/GFP and OPTN/PEX14-GFP fusion proteins retained on the GFP-Trap affinity matrix. The total signal intensities of the GFP proteins and PEX14 were both standardized to 100%. The bars represent the mean of 3 biological replicates. The densitometry quantifications of the relative normalized amounts (R-NA) of the proteins under study in the soluble extract input fractions are shown in the . c , e , Representative flow cytometry plots of each group (n = 2-3). The different colors represent the cell density at a given position. d , f , Quantification of the percentage of cells in the gated area. The data are shown as the mean ± SD and represent the values of 2-3 independent biological replicates. Relevant conditions were statistically compared (ns, non-significant; ***, p < 0.001).

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Cell Culture, Fluorescence, SDS Page, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: a , Structural model of the OPTN-OPTN-PEX14 complex before (left) and after (right) a 50 ns molecular dynamics (MD) simulation, depicted in either cartoon or surface representation. Residues were colored as follows: OPTN 420-510 in green (2x), and PEX14 in red (residues 1-137) and blue (residues 138-200). b , RMSD plot during MD simulations for the protein-protein complex.

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques:

Journal: bioRxiv

Article Title: PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

doi: 10.1101/2024.05.31.596776

Figure Lengend Snippet: a , Illustration of the multiple-sequence alignment coverage of the OPTN-OPTN-PEX14 sequence used as input for predicting the complex structure. The black line represents the total coverage for each residue. Residues corresponding to OPTN 420–510 (proteins A and B) and PEX14 1-200 (protein C) are indicated. b , Plot of the pLDDT scores for the top-ranked 5 structural models. Each model is depicted in a different color. c , Free energy landscape, d , RMSF, e , hydrogen bond, f , gyrate, and g , SASA changes during molecular dynamics simulations.

Article Snippet: The flippase (Flp) recombinase expression vector (pOG44; Thermo Fisher Scientific, V600520), the Flp-InTM inducible expression vector (pcDNA5/FRT/TO; Thermo Fisher Scientific, V652020), the lentiviral GFP expression vector (pLenti CMV GFP Puro; Addgene, plasmid 17448), and the plasmids encoding GFP (Clontech, 6085-1), the pOPINE GFP nanobody (Addgene, plasmid 49172 [ ]), non-tagged OPTN 1-577 (Origene, SC319257), OPTN 1-577 -GFP (Addgene, plasmid 27052 [ ]), OPTN 1-209 -GFP (Addgene, plasmid #68839 [ ]), OPTN 217-577 -GFP (Addgene, plasmid #68841 [ ]), or OPTN D474N -GFP (Addgene, plasmid #68847 [ ]) were commercially obtained.

Techniques: Sequencing, Residue

Journal: Scientific Reports

Article Title: Gene activated adipose tissue fragments as advanced autologous biomaterials for bone regeneration: osteogenic differentiation within the tissue and implications for clinical translation

doi: 10.1038/s41598-018-36283-6

Figure Lengend Snippet: Quantitative comparisons of bone marker gene expressions between MN (normal medium), MO (osteogenic medium) and MO + Ad.hBMP-2 groups. ALP ( A ), RUNX-2 ( B ), OPN ( C ), OCN ( D ) and BSP ( E ) gene expressions were significantly increased in MO + Ad.hBMP-2 group when compared to MO and MN, whilst significant differences were also found between MO and MN at most time points. h BMP-2 ( F ) was only expressed in Ad.hBMP-2 transduced group during the culture period, of which the highest gene expression was at 2 weeks. Four tissue discs cultured in each well were harvested as one sample for RNA isolation, and 9 samples per group were used for qRT-PCR. The significance level of MO + Ad.hBMP-2 vs . MN as well as MO vs . MN, was marked on the top of column without capped line (*for p < 0.05, **for p < 0.01 and ***for p < 0.001).

Article Snippet: After washing, rabbit anti -rat primary antibodies for OCN, OPN and Scl (Biorbyt) were applied to the sections at a dilution of 1:100 and left to incubate for 1 h at RT.

Techniques: Marker, Gene Expression, Cell Culture, Isolation, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Gene activated adipose tissue fragments as advanced autologous biomaterials for bone regeneration: osteogenic differentiation within the tissue and implications for clinical translation

doi: 10.1038/s41598-018-36283-6

Figure Lengend Snippet: Histological staining of untreated fresh adipose tissue discs. ( A ) (×2.5); ( B ) (×15): HE staining showing the structure of adipose tissue consisting of adipocytes (green arrow) and stromal vascular fraction (SVF) (red and blue arrows), which contains vascular tubes (red arrows) and a cellular complex including pre-adipocytes, mesenchymal stem cells, endothelial progenitor cells etc. (blue arrows). ( C ) Immunofluorescence of hBMP-2 revealing hBMP-2 was undetectable in untreated rat adipose tissue. ( D ) Alizarin red S staining showing calcium deposition was absent in untreated fresh adipose tissue. Immunohistochemistry of OCN ( E ), OPN ( F ) and Scl ( G ) indicating the co-localization of minimally endogenously expressed proteins of OCN, OPN and Scl in fresh adipose tissue, respectively.

Article Snippet: After washing, rabbit anti -rat primary antibodies for OCN, OPN and Scl (Biorbyt) were applied to the sections at a dilution of 1:100 and left to incubate for 1 h at RT.

Techniques: Staining, Immunofluorescence, Immunohistochemistry

Journal: Scientific Reports

Article Title: Gene activated adipose tissue fragments as advanced autologous biomaterials for bone regeneration: osteogenic differentiation within the tissue and implications for clinical translation

doi: 10.1038/s41598-018-36283-6

Figure Lengend Snippet: Immunohistochemical staining of sclerostin (Scl) and morphometric analysis of the IHC staining products: OCN, OPN and Scl. MN: ( A ) 1 week, ( B ) 2 weeks, ( C ) 4 weeks; MO: ( D ) 1 week, ( E ) 2 weeks, ( F ) 4 weeks; and MO + Ad.hBMP-2: ( G ) 1 week, ( H ) 2 weeks, ( I ) 4 weeks. The most intensive staining was presented in MO + Ad.hBMP-2 group at each time point. ( J ) Morphometric analysis of the IHC staining products: OCN, OPN and Scl at four weeks. The largest staining areas of OCN, OPN and Scl were observed in the sections from MO + Ad.hBMP-2 group, followed by MO, whilst MN the smallest. One representative section per tissue sample was analyzed. Nine tissue samples per group/condition were used for morphometric analysis. The significance level of MO + Ad.hBMP-2 vs . MN as well as MO vs . MN, was marked on the top of column without capped line (*for p < 0.05, **for p < 0.01 and ***for p < 0.001).

Article Snippet: After washing, rabbit anti -rat primary antibodies for OCN, OPN and Scl (Biorbyt) were applied to the sections at a dilution of 1:100 and left to incubate for 1 h at RT.

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry