1 ap rrid ab 2093492  (Proteintech)

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) HDAC6- or DYNC1I1-depleted A549 cells were infected with IAV at a multiplicity of infection (MOI) of 0.1 for 4 hours (h). NS1 and NP mRNA levels were quantified by RT-PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and expressed relative to siCtrl cells. ( B ) Cells in (A) were infected for 8 hours before WB with anti HDAC6, DYNC1I1, NP, and β-actin antibodies. NP levels were quantified relative to siCtrl cells. ( C ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 200 nM Baf, 1 μM Noc, or 10 μM CilioD before WB analysis using anti-NP and anti-GAPDH antibodies. ( D ) NP levels in (C), relative to untreated cells (Ctrl). ( E ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 10 μM CilioD, 1 μM Noc, 0.1 μM LatA, or 1 μM Noc and 0.1 μM LatA simultaneously before WB analysis with anti-NP and anti-GAPDH antibodies. ( F ) NP levels in (E), relative to untreated cells (Ctrl). ( G ) The cytoplasm entry assay was carried out in siCtrl-, siHDAC6-, or siDYNC1I1-treated sHeLa cells, which were then processed for IF with anti-M1 and anti-LAMP1 antibodies. Baf (200 nM) was used under the siCtrl condition to block IAV cytoplasm entry. Insets highlight M1-positive LAMP1 puncta. Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( H to J ) Quantification of the cell percentage with dispersed M1 (H), the number of M1 puncta per cell (I), and the percentage of M1-positive LAMP1 puncta (J) in (G). Error bars represent SDs [ n = 3 in (A), (B), (D), and (F); n = 10 in (H) to (J), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Microscopy

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) sHeLa cells treated as in in the presence or absence of Baf were processed for IF with anti-M1 and anti-LC3 antibodies. Insets highlight the colocalization between LC3 and M1, and white arrowheads point to colocalization (in untreated cells) and noncolocalization (in Baf-treated cells). Images were collected using a DeltaVision microscope. Scale bars, 5 μm. ( B ) Quantification of the LC3-positive M1 puncta in (A). Error bars represent SDs. ( C ) IAV cytoplasm entry in sHeLa cells was carried out as in , except that IAV was at an MOI of 30. Baf (200 nM) was used to block IAV fusion at LEs. Cell extracts were subjected to IP with LC3 antibodies before separating the coisolated proteins and WB analysis with anti-LC3, NP, and immunoglobulin G (ΙgG) (control) antibodies. ( D ) Quantification of the NP bound to LC3 in (C), expressed relative to infected cells not treated with Baf. ( E ) Cell extracts from sHeLa cells treated and processed as in (C). WB membranes were probed with anti-LC3, HDAC6, DYNC1I1, KIF5B, and ΙgG antibodies (control). ( F ) Quantification of the DYNC1I1 bound to LC3s in (E), expressed relative to mock-treated cells. Error bars represent SDs [ n = 3 in (D) and (F); n = 3 in (B), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Microscopy, Blocking Assay, Control, Infection

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) Working flow that led to the identification of PCNT as a factor in IAV cytoplasm entry. ( B ) DYNC1I1-, CDK1-, PCNT-, or PPP1CC-depleted sHeLa cells were infected with IAV at an MOI of 10 and processed for IF with the anti-M1 antibody at 3 hpi as in . Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( C and D ) Quantification of both the percentage of cells with dispersed M1 (C) and the amount of M1 puncta per cell (D) in (B). ( E ) sHeLa cells were transfected with siCtrl, siPCNT, or siDYNC1I1 for 48 hours, infected, and processed for ExM as in . Baf (200 nM) was used to block IAV fusion at LEs in sHeLa cells. Scale bars, ~4.5 μm (maximum projection images) and ~0.2 μm (inset images). ( F ) Quantification of the distance of luminal M1 puncta from the LAMP1-positive LE membrane in single slices of expanded cells shown in (E). ( G ) DYNC1I1- or PCNT-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 8 hours. Cell extracts were examined by WB with anti-NP, M2, PCNT, DYNC1 I1, and β-actin antibodies. ( H ) NP and M2 level quantification in (G). Bars represent average amounts relative to infected cells treated with siCtrl. ( I ) PCNT-depleted sHeLa cells were infected with luc-HSV-1 or luc-VaV at an MOI of 1 for 6 hours. Luciferase activity in cell extracts was then measured. Data represent the average luciferase activities expressed relative to the siCtrl for each virus. Error bars represent SDs [ n = 10 in (C) and (D), 50 cells counted per repeat; n = 3 in (F), (H), and (I)]. Asterisks indicate significant differences. h, hours.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Microscopy, Transfection, Blocking Assay, Membrane, Luciferase, Activity Assay, Virus

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 200 nM Baf or 100 nM Ctn before WB with the indicated antibodies. ( B ) NP levels in (A) relative to siCtrl. ( C ) PCNT-depleted sHeLa cells transfected with the GFP-PCNTB or GFP-PCNTS plasmid were infected with IAV at an MOI of 0.1 for 8 hours and examined by WB with the indicated antibodies. NP levels are relative to siCtrl. The GFP antibody was used for GFP-PCNTS. The PCNT antibody only detects PCNTB. ( D ) Cells as in (C) were infected with WSN-luc IAV for 16 hours, and luciferase activity was measured relative to infected siCtrl cells. ( E ) PCNT-depleted sHeLa cells were transfected with the GFP-PCNTS or GFP-PCNTS ΔPACT plasmid before IAV infection at an MOI of 0.1 for 8 hours and examined by WB analysis with the indicated antibodies. NP levels are relative to siCtrl. ( F ) Cells as in (E) were infected with WSN-luc IAV for 16 hours, and luciferase activity was measured relative to siCtrl. ( G ) DYNC1I1-, PCNT-, or LC3/PCNT-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 4 hours, and NP and NS1 mRNA levels were quantified by RT-PCR, normalized to GAPDH, and expressed relative to siCtrl. ( H ) Cells from (G) were infected with IAV for 8 hours before WB with the indicated antibodies. NP levels are relative to siCtrl. ( I ) DYNC1I1-, PCNT-, HDAC6-, or PCNT/HDAC6-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 4 hours, and NP and NS1 mRNA levels were quantified as in (G). ( J ) Cells as in (I) were infected for 8 hours before WB with the indicated antibodies. NP levels are relative to siCtrl. Error bars represent the SDs [ n = 3 in (B) to (J)]. Asterisks indicate significant differences. h, hours.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) PCNT-depleted atg7 −/− cells were infected with IAV at MOI 30 for 3 hours, and cell extracts were subjected to IP with an anti-LC3 antibody before examining the input and the coisolated proteins by WB with anti-LC3, NP, PCNT, DYNC1I1, and ΙgG (control) antibodies. ( B ) DYNC1I1, NP, and PCNT bound to LC3s in (A) relative to the infected siCtrl cells. ( C ) sHeLa APEX2KI and LC3 APEX2KI cells were infected with IAV as in , but 500 μM biotin phenol (BP) and 1 mM H 2 O 2 were added 30 and 1 min, respectively, before isolating biotinylated proteins. sHeLa APEX2KI cells without BP incubation were used as a negative control. The input and the affinity-purified proteins were analyzed by WB with antibodies against biotin, NP, PCNT, DYNC1I1, or β-actin. ( D and E ) Biotinylated DYNC1I1 (D) and NP (E) in (C) relative to the noninfected sHeLa APEX2KI cells. ( F ) PCNT-depleted atg7 −/− cells were processed for IF as in with antibodies against M1 and LC3. Insets highlight colocalization between M1 and LC3. Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( G ) Percentage of the LC3-positive M1 puncta in (F). ( H ) Model for IAV host cytoplasm entry. The lower pH of LEs triggers the fusion between endocytoses IAV VPs at LEs. Uncoating and cytoplasmic vRNP release is mediated by two dynein-dependent systems that take advantage of the pulling force of MT-based motors. vRNPs are linked to dynein motors via the LC3-PCNT adaptor complex or HDAC6, which binds ubiquitin. It is unknown which vRNP components interact with LC3s and HDAC6. Error bars represent SDs [ n = 3 in (B), (D), and (E); n = 5 in (C), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Control, Incubation, Negative Control, Affinity Purification, Microscopy, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Axon Trafficking Counteracts Aberrant Protein Aggregation in Neurons

doi: 10.1101/2025.01.16.633295

Figure Lengend Snippet: (A) Venn diagram showing four shared interactors between TIA1 and DIC1B interactomes (DYNC1i1 in BioGrid). (B, D) Mass spectrometry analysis of proteins interacting with GST-TIA1 (B) or GST-ANXA7 (D) in rat brain lysates, using GST tag as a control. Red dots indicate significantly enhanced interactors ( p < 0.05 and Log2 Fold Change > 1.2). Data from three replicates. (C, E) Immunoblots of ANXA7 in proteins pulled down by GST-TIA1 (C) ; TIA1 and DIC1B in proteins pulled down by GST-ANXA7 (E) from rat brain. (F) GO and KEGG pathway analysis of GST-ANXA7 interactors, including Biological Processes (BP), Cellular Components (CC), and Molecular Functions (MF). (G) Co-IP showing Myc-ANXA7 and HA-DIC1B interact with Flag-TIA1 in HEK293T cells. (H) Confocal images of endogenous TIA1 (green) and ANXA7 or DIC1B (red) in the cortex and hippocampus of P34 mouse brain. Scale bars = 500 μm and 10 μm. (I) Time-lapse images showing retrograde co-trafficking of light-induced Opto-TIA1 (red) and ANXA7-GFP (green) granules in DIV9 rat hippocampal neurons. Scale bar = 2 µm. Arrowheads indicate co-trafficking. (J) In vitro protein pull-down assay schematic. (K) Purified Myc-ANXA7 protein enhances TIA1 and Flag-DIC interaction, shown by increased TIA1 co-IP’d with Flag-DIC. (L) Co-IP assay examining the interaction between endogenous DIC1B and HA-tagged TIA1 using anti-HA magnetic beads in cultured DIV11 rat cortical neurons. The interaction is studied under endogenous ANXA7 knockdown (shANXA7) or Myc-ANXA7 overexpression conditions. (M) Quantifying TIA1-DIC1B interaction from (L) shows the effects of different ANXA7 levels (n = 5, 4, 4). (N) Schematic diagram of FLIM-FRET to examine the affinity between GFP-TIA1 (donor) and DIC1B-mRFP (acceptor) under varying levels of ANXA7. (O) Represented images showing colour-coded GFP-TIA1 lifetime in axon shafts of transfected neurons, with lifetime (P) and FRET efficiency (Q) quantified and compared across indicated groups. Scale bars =2 μm (n = 29, 37, 35, 49). Data represent mean ± SEM; in (M) two-tailed unpaired t -test; in (P, Q) one-way ANOVA. * p <0.05, *** p <0.001, ns non-significant.

Article Snippet: Primary antibodies: anti-TIA1 (#sc-166247, Santa Cruz), anti-TIA1 (#12133-2-AP, Proteintech), anti-SQSTM1/p62 (#A11247, ABclonal), anti-G3BP1 (#sc-365338, Santa Cruz), anti-Rab5 (#3547, Cell Signaling Technology), anti-LC3B (#83506, Cell Signaling Technology), anti-LAMP1 (#ab13523, Abcam), anti-DYNC1I1 (#13808-1-AP, Proteintech), anti-ANXA7 (#10154-2-AP, Proteintech), anti-GAPDH (#10494-1-AP, Proteintech), anti-HA (#3724, Cell Signaling Technology), anti-Myc (#60003-2-Ig, Proteintech), anti-Myc (#16286-1-AP, Proteintech), anti-Flag (#20543-1-AP, Proteintech), anti-TDP-43 (#89789, Cell Signaling Technology), anti-Iba1 (#019-19741, FUJIFILM).

Techniques: Mass Spectrometry, Control, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Pull Down Assay, Purification, Magnetic Beads, Cell Culture, Knockdown, Over Expression, Transfection, Two Tailed Test

Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 3. PGC-1α increases the levels of mitochondrial transport proteins in the cortex of APP/PS1 mice. Immunohistochemistry was used to examine the expression patterns and levels of (Aa-a’) MFN2, (Ba-a’) KIF5A, and (Ca-a’) Dync1i1 in cortex samples from WT/2 × Tg-AD mice. The impact of treatment (AAV-Vector/AAV-PGC-1α) on the expression and levels of (Ab-b’) MFN2, (Bb- b’) KIF5A, and (Cb-b’) Dync1i1 in cortex samples from 2 × Tg-AD mice was also assessed using immunohistochemistry. Scale bars = 100 μm. The expression patterns and qualification of (Bc-c’) KIF5A in cortical samples from 2 × Tg-AD mice treated with AAV-Vector/AAV-PGC-1α were examined with immunoflu orescence. Scale bars = 200 μm. Green = HA-labeled PGC-1α; Red = KIF5A; Blue = DAPI. (Bd-d’) N2A cells were transfected with pEnCMV/PGC-1α plasmid and plasmid-encoding APPswe for 48 h. The expression patterns and quantifi cation of KIF5A in the cells were studied with western blot. For each group, n = 6. Values are expressed as the means ± S.E.M. Significance levels were set at ** p < 0.01 and *** p < 0.001 for noted differences between AAV-Vector- and AAV-PGC-1α-infused AD mice or pEnCMV- or PGC-1α-transfected APPswe cells. GAPDH was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Immunohistochemistry, Expressing, Plasmid Preparation, Labeling, Transfection, Western Blot, Control

Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 5. RID3, but not RID2, of the PGC-1α region is indispensable for promoting retrograde transport of axonal mitochondria and enhancing mitochondrial auto phagic clearance. N2A cells were co-transfected with plasmid encoding APPswe and pEnCMV/PGC-1α/PGC-1αmL2/PGC-1αmL3 plasmids. The lysates were subjected to immunoblotting using the specified antibodies. Expression patterns and quantification of (A) the retrograde transport protein Dync1i1 and (B) mitophagy-relevant proteins, including (a) Parkin, (b) Pink, (c) LC3-I/II, (d) Beclin and (e) P62, were examined via western blot analysis. GAPDH was utilized as a loading control. Each group consisted of n = 6 samples. (C) Fluorescence confocal microscopy was employed to detect JC-1 signals in N2A cells. Data represent three independent measurements. Fluorescence was captured using excitation at 488 nm and adjusting the emission of confocal microscopy for J-monomers (visible as green) and J- aggregates (visible as red/orange). (D) The ratios of J-aggregates to J-monomers were quantified as an indicator of mitochondrial membrane potential (MMP). Each group included n = 6 samples. Scale bars = 10 μm. The data underwent One-way ANOVA analysis to assess the effects of individual factors in isolation from others. Different letters indicate significant differences in mean values between groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, Fluorescence, Confocal Microscopy, Membrane, Isolation

Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 3. PGC-1α increases the levels of mitochondrial transport proteins in the cortex of APP/PS1 mice. Immunohistochemistry was used to examine the expression patterns and levels of (Aa-a’) MFN2, (Ba-a’) KIF5A, and (Ca-a’) Dync1i1 in cortex samples from WT/2 × Tg-AD mice. The impact of treatment (AAV-Vector/AAV-PGC-1α) on the expression and levels of (Ab-b’) MFN2, (Bb- b’) KIF5A, and (Cb-b’) Dync1i1 in cortex samples from 2 × Tg-AD mice was also assessed using immunohistochemistry. Scale bars = 100 μm. The expression patterns and qualification of (Bc-c’) KIF5A in cortical samples from 2 × Tg-AD mice treated with AAV-Vector/AAV-PGC-1α were examined with immunoflu orescence. Scale bars = 200 μm. Green = HA-labeled PGC-1α; Red = KIF5A; Blue = DAPI. (Bd-d’) N2A cells were transfected with pEnCMV/PGC-1α plasmid and plasmid-encoding APPswe for 48 h. The expression patterns and quantifi cation of KIF5A in the cells were studied with western blot. For each group, n = 6. Values are expressed as the means ± S.E.M. Significance levels were set at ** p < 0.01 and *** p < 0.001 for noted differences between AAV-Vector- and AAV-PGC-1α-infused AD mice or pEnCMV- or PGC-1α-transfected APPswe cells. GAPDH was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Immunohistochemistry, Expressing, Plasmid Preparation, Labeling, Transfection, Western Blot, Control

Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 5. RID3, but not RID2, of the PGC-1α region is indispensable for promoting retrograde transport of axonal mitochondria and enhancing mitochondrial auto phagic clearance. N2A cells were co-transfected with plasmid encoding APPswe and pEnCMV/PGC-1α/PGC-1αmL2/PGC-1αmL3 plasmids. The lysates were subjected to immunoblotting using the specified antibodies. Expression patterns and quantification of (A) the retrograde transport protein Dync1i1 and (B) mitophagy-relevant proteins, including (a) Parkin, (b) Pink, (c) LC3-I/II, (d) Beclin and (e) P62, were examined via western blot analysis. GAPDH was utilized as a loading control. Each group consisted of n = 6 samples. (C) Fluorescence confocal microscopy was employed to detect JC-1 signals in N2A cells. Data represent three independent measurements. Fluorescence was captured using excitation at 488 nm and adjusting the emission of confocal microscopy for J-monomers (visible as green) and J- aggregates (visible as red/orange). (D) The ratios of J-aggregates to J-monomers were quantified as an indicator of mitochondrial membrane potential (MMP). Each group included n = 6 samples. Scale bars = 10 μm. The data underwent One-way ANOVA analysis to assess the effects of individual factors in isolation from others. Different letters indicate significant differences in mean values between groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, Fluorescence, Confocal Microscopy, Membrane, Isolation

Journal: Neurobiology of disease

Article Title: Pain sensitivity related to gamma oscillation of parvalbumin interneuron in primary somatosensory cortex in Dync1i1 -/- mice.

doi: 10.1016/j.nbd.2023.106170

Figure Lengend Snippet: Fig. 1. No anxiety or gait change in Dync1i1 knockout mice. (A). Crossbreeding scheme in order to obtain Dync1i1−/−mice and their littermates WT mice. (B). Representative genotyping PCR results of Dync1i1−/−mice, Dync1i1+/−mice and WT mice. (C). The expression of Dync1i1 protein in the cortex of Dync1i1−/−mice and WT mice. (E). In the elevated plus maze (EPM), the Dync1i1−/−mice spent the same amount of time in closed arms and open arms (n = 7 for Dync1i1−/−mice, n = 11 for WT mice，n.s., not significant unpaired t-test. (F). Also, the same frequency of entries was made into both open and closed arms (n = 7 for Dync1i1−/−mice, n = 11 for WT mice，n.s., not significant unpaired t-test. (G–J). According to the footprint analysis performed using the CatWalk system, Dync1i1−/−mice and their WT littermates exhibited no significant difference in intensity (G), speed (H), time (I), and stride length (J) to the walkway (n = 6 for each group，n.s., not significant unpaired t-test).

Article Snippet: We blocked the membrane with 5% BSA in TBST buffer (10 mmol/L Tris, pH 7.5; 100 mmol/L NaCl; and 0.1% Tween 20), and was incubated overnight at 4◦C with Dync1i1 (Proteintech, USA) primary antibodies (1:1000 dilution in 5% BSA in TBST buffer) .On the second day, the membrane was incubated with anti-rabbit secondary antibodies (1:5000; Invitrogen, USA) and target strips were then imaged using a chemiluminescence kit (Beyotime Biotechnology, China).

Techniques: Knock-Out, Expressing

Journal: Neurobiology of disease

Article Title: Pain sensitivity related to gamma oscillation of parvalbumin interneuron in primary somatosensory cortex in Dync1i1 -/- mice.

doi: 10.1016/j.nbd.2023.106170

Figure Lengend Snippet: Fig. 2. Dync1i1−/−mice showed altered pain threshold in pain-related behavioral tests (A). Experimental procedure diagram of pain behavior tests in mice. (B). Paw withdrawal rates to graded von Frey stimulation of the hind paw between Dync1i1−/−mice and their WT littermates (n = 6 for each group; *P < 0.05 two-way ANOVA with multiple comparisons). (C). Paw withdrawal latency to hotplate experiment between Dync1i1−/−mice and their WT littermates (n = 8 for Dync1i1−/−mice, n = 9 for WT mice; *P < 0.037 unpaired t-test). (D). Paw withdrawal latency to acetone test between Dync1i1−/−mice and their WT littermates (n = 7 for each group; *P < 0.0244 unpaired t-test). (E). Paw withdrawal rate to brush test between Dync1i1−/−mice and their WT littermates (n = 8 for each group; n.s. not significant). (F). Typical changes and quantification of C-Fos expression in S1HL (top), ACC (middle), and S2 (bottom) following Von Frey stimulation between Dync1i1−/−mice and their WT littermates. (n = 5 for each group *P < 0.05 n.s., not significant).

Article Snippet: We blocked the membrane with 5% BSA in TBST buffer (10 mmol/L Tris, pH 7.5; 100 mmol/L NaCl; and 0.1% Tween 20), and was incubated overnight at 4◦C with Dync1i1 (Proteintech, USA) primary antibodies (1:1000 dilution in 5% BSA in TBST buffer) .On the second day, the membrane was incubated with anti-rabbit secondary antibodies (1:5000; Invitrogen, USA) and target strips were then imaged using a chemiluminescence kit (Beyotime Biotechnology, China).

Techniques: Expressing

Journal: Neurobiology of disease

Article Title: Pain sensitivity related to gamma oscillation of parvalbumin interneuron in primary somatosensory cortex in Dync1i1 -/- mice.

doi: 10.1016/j.nbd.2023.106170

Figure Lengend Snippet: Fig. 3. Abnormal power of multi-frequency oscillations in Dync1i1−/−mice. (A). LFP signals from mice with Dync1i1−/−(left) and their WT littermates (right) as well as their filtered theta, alpha, and beta components. (B). Power spectrogram of LFP from Dync1i1−/−(left) and their WT littermates (right) (C). Top: Power spectral density of LFP from 0 to 100 Hz in Dync1i1−/−mice and their WT littermates (n = 5 for each group). Bottom: Power spectral density of LFP from 0 to 30 Hz in Dync1i1−/−and their WT littermates (n = 5 for each group). (D). LFP signals power spectral density in the theta (4–8 Hz), alpha (8–12 Hz), beta (15–29 Hz), low gamma (30–60 Hz), and high gamma (60–100 Hz) (n.s., not significant *P < 0.05, Unpaired t-test, n = 5 for each group.)

Article Snippet: We blocked the membrane with 5% BSA in TBST buffer (10 mmol/L Tris, pH 7.5; 100 mmol/L NaCl; and 0.1% Tween 20), and was incubated overnight at 4◦C with Dync1i1 (Proteintech, USA) primary antibodies (1:1000 dilution in 5% BSA in TBST buffer) .On the second day, the membrane was incubated with anti-rabbit secondary antibodies (1:5000; Invitrogen, USA) and target strips were then imaged using a chemiluminescence kit (Beyotime Biotechnology, China).

Techniques:

Journal: Neurobiology of disease

Article Title: Pain sensitivity related to gamma oscillation of parvalbumin interneuron in primary somatosensory cortex in Dync1i1 -/- mice.

doi: 10.1016/j.nbd.2023.106170

Figure Lengend Snippet: Fig. 4. Dync1i1−/−mice exhibit abnormal gamma oscillations in the S1 hindlimb cortex (S1HL) in conjunction with nociceptive behavior. (A). Left: Time-frequency representative distributions in S1HL for all trials with withdrawal response to 2 g von Frey stimulation of the contralateral hind paw in Dync1i1−/−mice (n = 5). Right: Time-frequency representative distributions of the same conditioning withdrawal trials in WT mice (n = 5) (B). Averaged different gamma band oscillations' time-frequency responses elicited by Von Frey stimulation in Dync1i1−/−mice and their WT littermates. Left: low gamma (F (1, 288) = 157, *P < 0.05). Right: high gamma(F (1, 288) = 104.6, *P < 0.05). (C). Analysis of the time-frequency representations in with and without paw withdrawals for different frequency bands 1 s after application of filaments of 2 g. For each animal, data were averaged across all withdrawal and no-withdrawal trials in 50 ms time bins at the specified frequency ranges including Top: theta (F (1, 930) = 115.4, *P < 0.05), alpha (F (1, 930) = 53.35, *P < 0.05), beta(F (1, 930) = 108.1, *P < 0.05), Low gamma (F (1, 930) = 1.1, P = 0.2946) and High gamma (F (1, 930) =1.309, P = 0.2529) in Dync1i −/−mice (n = 5, *P < 0.05 two-way repeated measures ANOVA). Bottom: Theta (F (1, 930) = 31.24, *P < 0.05), alpha (F(1,930) = 3.179, P = 0.0749, beta (F (1, 930) = 43.25, *P < 0.05), Low gamma (F (1, 930) = 59.9, *P < 0.05) and High gamma (F (1,930) = 57.24, *P < 0.05) in WT mice (n = 5; *P < 0.05 two-way repeated measures ANOVA).

Article Snippet: We blocked the membrane with 5% BSA in TBST buffer (10 mmol/L Tris, pH 7.5; 100 mmol/L NaCl; and 0.1% Tween 20), and was incubated overnight at 4◦C with Dync1i1 (Proteintech, USA) primary antibodies (1:1000 dilution in 5% BSA in TBST buffer) .On the second day, the membrane was incubated with anti-rabbit secondary antibodies (1:5000; Invitrogen, USA) and target strips were then imaged using a chemiluminescence kit (Beyotime Biotechnology, China).

Techniques:

Journal: Neurobiology of disease

Article Title: Pain sensitivity related to gamma oscillation of parvalbumin interneuron in primary somatosensory cortex in Dync1i1 -/- mice.

doi: 10.1016/j.nbd.2023.106170

Figure Lengend Snippet: Fig. 5. Changes in putative interneurons and pyramidal neurons in Dync1i1−/−and their WT littermates. (A). Typical spike responses of excitatory responses (left), lack of responses (middle), and inhibitory responses (right) to Von Frey stimulation. (B, C) A comparison of the percentages of neurons that respond differently to nociceptive stimulation in Dync1i1−/−mice and their WT littermates (B, interneurons; C, pyramidal neurons). Excitatory, lack of, and inhibitory responses are coded in orange, gray, and green, respectively. The proportion of putative interneurons and pyramidal neurons shows no difference in Excitatory, lack of, and inhibitory responses between Dync1i1−/−and their WT littermates (putative interneurons, n = 44 for Dync1i1−/−, n = 56 for WT littermates; n.s. not significant P = 0.6263 Chi- square, putative pyramidal neurons, n = 83 for Dync1i1−/−, n = 112 for WT littermates n = 3 mice for each group. n.s. Not significant. P = 0.7446 Chi-square). (D, E). Representative firing rates of recorded modulated units including (D) Dync1i1−/−mice and (E) WT mice before and after the 2 g Von Frey stimulation onset (A normalized firing rate is calculated by dividing the maximum firing rate by firing rate of each modulated unit). Colour codes are used to indicate low (blue) to high (red) firing activity. Units are plotted separately according to the direction of modulation, from stimulus-induced increase (left) to decrease (right) of the firing rate. (F). Average firing rates of neurons recorded before and after application of filaments of 2 g (putative interneurons and putative pyramidal neurons). Each neuron's values have been normalized (Norm.) to its maximum firing rate. Top inset: CV before and after application of filaments of 2 g for Dync1i1−/−and their WT lit termates. Note that the lower CV in putative interneurons for Dync1i1−/−mice (*P < 0.05; Unpaired t-test). CV did not differ between WT and Dync1i1−/−mice in putative pyramidal neurons.(n.s. not significant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: We blocked the membrane with 5% BSA in TBST buffer (10 mmol/L Tris, pH 7.5; 100 mmol/L NaCl; and 0.1% Tween 20), and was incubated overnight at 4◦C with Dync1i1 (Proteintech, USA) primary antibodies (1:1000 dilution in 5% BSA in TBST buffer) .On the second day, the membrane was incubated with anti-rabbit secondary antibodies (1:5000; Invitrogen, USA) and target strips were then imaged using a chemiluminescence kit (Beyotime Biotechnology, China).

Techniques: Comparison, Activity Assay

Journal: Neurobiology of disease

Article Title: Pain sensitivity related to gamma oscillation of parvalbumin interneuron in primary somatosensory cortex in Dync1i1 -/- mice.

doi: 10.1016/j.nbd.2023.106170

Figure Lengend Snippet: Fig. 6. Impaired S1HL firing dynamics in Dync1i1−/−mice. (A, B) left: The coherence between broadband frequency and putative interneurons' spikes were measured in S1HL before and after 2 g von Frey stimulation of the contralateral hind paw in Dync1i1−/−(A) and their WT littermates (B). Right: coherence values between gamma band oscillation and putative interneurons' spikes were similar before and after in S1HL in Dync1i1−/−mice (n.s. not significant). (A). Coherence values between high gamma band oscillation and putative interneurons' spikes were larger post-stimulate than pre-stimulate in WT mice (*P < 0.05 paired t-test), and coherence values between low gamma band and putative interneurons' spikes were similar before and after in contralateral S1HL in WT mice (n.s. not significant) (B). (C, D). Left: The coherence between broadband frequency and putative pyramidal neurons were measured in S1HL before and after 2 g von Frey stimulation of the contralateral hind paw in Dync1i1−/−(C) and their WT littermates (D). Right: Coherence values between gamma band oscillation and putative pyramidal neurons was larger post-stimulate than pre-stimulate in Dync1i1−/−mice (C) (*P < 0.05 paired t-test). Coherence values between gamma band oscillation and putative pyramidal neurons' spikes were similar before and after in S1HL in WT littermates (n.s. not significant) (D). (E, F). left: Representative polar plots of putative in terneurons' spikes distributions along the gamma phase before and after 2 g Von Frey stimulate onset in WT mice (E) and in Dync1i1−/−(F). Right: MVL values for both WT (E) and Dync1i1−/−mice (F) before and after Von Frey stimulation (n.s. not significant *P < 0.05 paired t-test).

Article Snippet: We blocked the membrane with 5% BSA in TBST buffer (10 mmol/L Tris, pH 7.5; 100 mmol/L NaCl; and 0.1% Tween 20), and was incubated overnight at 4◦C with Dync1i1 (Proteintech, USA) primary antibodies (1:1000 dilution in 5% BSA in TBST buffer) .On the second day, the membrane was incubated with anti-rabbit secondary antibodies (1:5000; Invitrogen, USA) and target strips were then imaged using a chemiluminescence kit (Beyotime Biotechnology, China).

Techniques:

Journal: Neurobiology of disease

Article Title: Pain sensitivity related to gamma oscillation of parvalbumin interneuron in primary somatosensory cortex in Dync1i1 -/- mice.

doi: 10.1016/j.nbd.2023.106170

Figure Lengend Snippet: Fig. 7. Optogenetic activation and CNO Inhibition of S1HL interneurons in Von Frey test. (A) Experimental procedure diagram. (B). Expression of ChR2-mCherry in the S1HL of a Dync1i1−/−mouse. Scale bar represents 200 μm. (C). LFP bandpass filtered between 30 Hz and 60 Hz on Dync1i1−/−mice in the absence and presence of laser illumination at 40 Hz. (D). The representation of time-frequency power during an illumination period of 3 s using 40 Hz laser pulses (Presented by dotted blue line) of PV-ChR2-mCherry mice (right) or control mice expressing mCherry alone in PV neurons (left) (n = 5 mice per group and 10 trials each). (E). Changes in mean power (The power is expressed as a percentage over an illumination period of 3 s at a frequency of 40 Hz normalized for a baseline period of 1 s before illumination) in theta (4–8 Hz), alpha (8–12 Hz), beta (15–29 Hz), low gamma (30–60 Hz), and high gamma (60–100 Hz) frequency bands in PV-mCherry versus PV-ChR2- mCherry animals (n = 3 in each group *P < 0.05 two-way ANOVA with multiple comparisons). (F, G). The percentage of withdrawal rate when 2 g Von Frey stimulation onset are shown at baseline and during 40 Hz illumination in Dync1i1−/−PV-ChR2-mCherry mice (F)(n = 6; *P < 0.05 paired t-test) and Dync1i1−/−PV- mCherry mice (G)(n = 7; n.s. not significant). (I). The percentage of withdrawal rate when 2 g Von Frey stimulation onset is shown at baseline and after application of CNO in 30 min in WT PV-hM4D-mCherry mice. (n = 7; *P < 0.05 paired t-test). (J). The percentage of withdrawal rate when 2 g Von Frey stimulation onset is shown at baseline and after application of saline in 30 min in WT PV-hM4D-mCherry mice (n = 7; n.s. not significant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: We blocked the membrane with 5% BSA in TBST buffer (10 mmol/L Tris, pH 7.5; 100 mmol/L NaCl; and 0.1% Tween 20), and was incubated overnight at 4◦C with Dync1i1 (Proteintech, USA) primary antibodies (1:1000 dilution in 5% BSA in TBST buffer) .On the second day, the membrane was incubated with anti-rabbit secondary antibodies (1:5000; Invitrogen, USA) and target strips were then imaged using a chemiluminescence kit (Beyotime Biotechnology, China).

Techniques: Activation Assay, Inhibition, Expressing, Control, Saline