Journal: bioRxiv

Article Title: SMPD3 -mediated extracellular vesicle biogenesis inhibits oligodendroglioma growth

doi: 10.1101/2020.07.14.202200

Figure Lengend Snippet: A,B. Scanning EM of BT054 (A) and BT088 (B) cells. Red arrows mark vesicles. C,C’. Transmission electron microscopy of BT088 EVs. Higher magnification image shown in (C’). D-G. Nanosight tracking analysis of BT054 and BT088 EVs isolated by sequential ultracentrifugation (D,E) and density gradient ultracentrifugation (fraction 4; F). CD9 + EVs analyzed using nanoscale flow cytometry (G). H-J. Western blots of BT088 whole-cell lysates (CL) and EV lysates obtained by sequential ultracentrifugation, analyzed for the expression of EV markers (Alix, CD9, Cetp, Flotillin1) (H) and Calreticulin, Calnexin (ER), GM130 (Golgi body), Vdac (mitochondria), and Pex5 (Peroxisomes) (I). Density gradient ultracentrifugation of BT088 EVs, with 8 fractions analysed by Western blots for Alix, CD9, Cetp, Calnexin, and Vdac. K-O. Schematic of pellet assay mixing BT088 cells expressing Cre-GFP at 5:1 ratio with NIH-3T3 cells expressing a dual BFP-loxP-dsRed reporter (K). Analysis of GFP (L,M), BFP (L,N) and dsRed (L,O) expression in pellets after 3 DIV. White arrows mark dsRed + BFP − GFP − cells. White arrowheads mark dsRed + BFP + GFP − cells. Scale bars: 2 μm in (A,B); 500 nm in (C); 50 nm in (C’).

Article Snippet: Primary antibodies included: Flotillin1 (Cell signalling; #3253), CD9 (Santa Crutz; #sc9148), nSMase2 (Abcam; #ab85017), Cetp (Abcam; #ab2726), Alix (Cell Signaling; #2171S), GM130 (BD Biosciences; #610822), Calnexin (Abcam; #ab22595), Calreticulin (Abcam; #ab2907), Pex5 (Novus Biologicals; #NBP1-87185), VDAC (Cell Signaling; #4661), and Actin (Abcam; #ab8227).

Techniques: Transmission Assay, Electron Microscopy, Isolation, Flow Cytometry, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b-deficient mice have altered atrial electrophysiology. ( A ) In vitro electrophysiological measurements from Langendorff-perfused hearts show an increase of atrial refractory period (ARP) and atrioventricular-nodal refractory period (AVNRP) in TRIP8b-deficient mice, without changes in sino-nodal activity (sino-nodal recovery time, SNRT) and heart rate (HR). ( B ) Representative tracings are shown for wild-type and TRIP8b-deficient mice. A, atrial activity; atrium electrophysiological tracings from the atrium; V, ventricular activity; ventricle electrophysiological tracings from the ventricle; black arrowheads mark atrial or ventricular stimulation. ( C ) Ganglionic blockade with 0.5 mM hexamethonium leads to a reduction of AVNRP in TRIP8b-deficient mice. Data are presented as box plots (minimum to maximum, n = 5–11 per genotype) and were compared using an unpaired t -test or Mann–Whitney, as appropriate.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: In Vitro, Activity Assay, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: Trip8b mRNA is present in the cardiac nervous system. ( A ) Exon 6–7 can be amplified in cDNA of ganglia-containing atrial tissue of wild-type but not of TRIP8b-deficient mice (left panel). Quantitative PCR analyses show that exon 8–9, 9–10, and 13–14 of Trip8b are still detectable in knockout mice. Data (normalized to Cdkn1b) are presented as individual data points with SEM ( n = 3, right panel) and were compared via one-way ANOVA followed by Sidaks’ multiple comparison test; ns, not significant. ( B ) Trip8b mRNA can be visualized with RNAscope in situ hybridization in neuronal cell bodies of cardiac ganglia. Black arrows in magnifications point to single neurons with Trip8b mRNAs. ( C ) Trip8b mRNA (black arrows) can be visualized with RNAscope in situ hybridization in cardiac nerves of wild-type mice.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Amplification, Real-time Polymerase Chain Reaction, Knock-Out, In Situ Hybridization

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: Trip8b mRNAs are detectable in the cardiac conduction system to a lower amount than in the intracardiac nervous system. ( A ) Sinus node and ( B ) atrioventricular node (AV node) were identified via hematoxylin and eosin (H&E) staining and Hcn4 RNAscope in situ hybridization. Subsequent sections treated with a probe specific for Trip8b show solitary mRNA spots (black arrows) surrounding the sinus node artery and in the AV node. Nuclei are counterstained with hematoxylin in blue. ( C ) The histogram shows the distribution of Trip8b mRNA spots per cell in the intracardiac nervous system (ICNS, nerves, and ganglia), sinus node, and AV node of wild-type and TRIP8b-deficient mice. Overall, 279–404 cells were analyzed for each region of interest per genotype, n = 2–3 images/genotype. ( D ) Trip8b in situ hybridization (red) detects mRNA in wild-type mice but also, to a lower amount, in knockout mice.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Staining, In Situ Hybridization, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b protein is not detectable in the atrial lysates and the cardiac autonomic nervous system. ( A ) Western blot analysis of brain tissue as positive control detects specific bands for TRIP8b (NBP2-38840, Novusbio) already at 2.5 µg total protein. For the heart, 50 µg atrial or ventricular lysate did not show any specific bands, while HCN4 ( B ) is detectable in both genotypes. ( C ) Immunohistochemistry for TRIP8b (APR-070, Alomone Labs) on paraffin sections was established in the central nervous system, more specifically, the cerebral cortex. Neurons positive for TRIP8b are detectable in the wild-type animals but not cortex of TRIP8b-deficient animals. ( D ) To increase the sensitivity of detection, atrial whole-mount preparations (upper panel shows exemplary staining with αTH ab152, Merck Millipore) were stained and ganglia cut out for confocal microscopy (bottom panel with αTH ab76442, Abcam). No specific signal was obtained for TRIP8b (APR-070, Alomone Labs), and no differences were detectable between the genotypes.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Western Blot, Positive Control, Immunohistochemistry, Staining, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b protein is not detectable in the cardiac conduction system in wild-type mice. Sinus node (upper panel) and atrioventricular node (AV node, bottom panel) were identified by anatomical landmarks and HCN4 staining (green). No staining for TRIP8b (red, APR-070, Alomone Labs) was detectable beyond the background.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: HCN channel expression in intracardiac ganglia. ( A ) In situ hybridization of two exemplary wild-type ganglia for Hcn2 (green) and Hcn4 (red). Both mRNAs are present within the ganglia. Boxed area is magnified in the inlay. ( B ) Gene expression analysis of Hcn2 and Hcn4 in TRIP8b-deficient mice and wild-type littermates. Data are presented as normalized gene expression to Cdkn1b using the formula 2 −ΔCt (box plots, minimum to maximum, n = 6 per genotype) and were compared using Mann–Whitney test.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Expressing, In Situ Hybridization, MANN-WHITNEY

Journal: bioRxiv

Article Title: Peroxisomal Dysfunction Drives Loss of Dynamic Nutrient Responses to Initiate Metabolic Inflexibility During Aging

doi: 10.1101/2025.02.14.638340

Figure Lengend Snippet: a , Fluorescent images showing the effect of PRX-5 loss on peroxisome import. RFP puncta become cytoplasmic upon induced PRX-5 loss. Scale bar = 10µm. b, Quantification of punctate peroxisomes upon inducible loss of PRX-5 protein. ( N=3 biological replicates, n=20 animals per condition, student’s two tailed t test) c, Magnified images of GFP labelled mitochondria in day 5 control and PRX-5 degraded adults showing gross alterations and mitochondrial swelling upon PRX-5 loss. White encircled mitochondria highlight common defects. Scale bar = 5 µm d, Quantification of by tetramethylrhodamine, ethyl ester (TMRE) intensity in two TMRE concentrations (10μM and 2μM) with and without 25μM FCCP treatment to establish that TMRE readily stains polarized mitochondria, and its intensity decreases with FCCP treatment. ( N=2 biological replicates, n=15-17 animals per condition, student’s two tailed t test). e-f, Representative fluorescent images and quantification showing auxin treatment alone does not affect mitochondrial polarization in WT animals. Scale bar = 10 µm ( N=2 biological replicates, n=15-17 animals per condition, student’s two tailed t test). g, Quantification of PEX5/Actin and MFN1/Actin expression in WT and PEX5-KO A549 cells. ( N=4 biological replicates, student’s two tailed t test). (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Taqman probes used to target each gene of interest are as follows: ACOX1: Hs01074240_m1, PEX5: Hs00165604_m1, PEX6: Hs01122110_m1, PEX10: Hs00538216_m1, PEX12: Hs01558848_g1, PEX13: Hs00968434_m1, PEX14: Hs00992867_m1, PEX19: Hs01005928_m1.

Techniques: Two Tailed Test, Control, Expressing

Journal: bioRxiv

Article Title: Peroxisomal Dysfunction Drives Loss of Dynamic Nutrient Responses to Initiate Metabolic Inflexibility During Aging

doi: 10.1101/2025.02.14.638340

Figure Lengend Snippet: a , Representative fluorescent images showing acute mitochondrial dynamic changes upon inducible peroxisomal import loss in transgenic line expressing vha-6p::mRFP-SKL;eft-3p::tomm-20(1-49aa)::GFP to label peroxisomes and mitochondria. Scale bar = 5 µm. (N=3 biological replicates). Arrows mark visible areas where peroxisome import loss phenotype is visible. b, Representative fluorescent images showing accelerated swelling of muscle mitochondria with age upon inducible peroxisomal import loss initiated at baseline, day 1 of adulthood and imaged at day 3, 5, and 8 of adulthood subsequently. Scale bar = 5 µm. (N=3 biological replicates). c-e, Quantification of mitochondrial dynamic parameters: count, area (μm 2 ), and circularity with age in animals with inducible peroxisome import loss initiated at day 1 of adulthood and imaged at day 3, 5, and 8 of adulthood subsequently (N=3 biological replicates, n=13-31 animals per condition, one way ANOVA). f-g, Representative fluorescent images and quantification showing mitochondrial hyperpolarization measured by tetramethylrhodamine, ethyl ester (TMRE) retention with age upon inducible peroxisomal import loss initiated at day 1 of adulthood. Images represent day 3 adulthood. Quantification provided over baseline (day 1), and day 3, day 5, and day 8 of adulthood. Scale bar = 10 µm. (N=3 biological replicates, n=14-21 animals per condition, one way ANOVA). h, Representative western blot showing PEX5 loss in A549 PEX5 KO cells relative to WT controls. (N=4 biological replicates). i, Pyruvate-dependent oxygen consumption rate (OCR) over mitochondrial stress test in WT and PEX5 KO cells show gross mitochondrial oxidative dysfunction in PEX5 KO cells. j-l, Quantification of basal, oligomycin inhibited, and FCCP induced maximal respiration over mitochondrial stress test in WT and PEX5 KO cells. (N=3 biological replicates, student’s two-tailed t test). m, glucose-dependent extracellular acidification rate (ECAR) in WT and PEX5 KO cells showing increased glycolytic reliance in PEX5 KO cells. (N=2 biological replicates, student’s two-tailed t test). n, peroxisomes regulate mitochondrial dynamics and bioenergetics through interorganelle regulation. Data are presented as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Taqman probes used to target each gene of interest are as follows: ACOX1: Hs01074240_m1, PEX5: Hs00165604_m1, PEX6: Hs01122110_m1, PEX10: Hs00538216_m1, PEX12: Hs01558848_g1, PEX13: Hs00968434_m1, PEX14: Hs00992867_m1, PEX19: Hs01005928_m1.

Techniques: Transgenic Assay, Expressing, Western Blot, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Functional multi-organelle units control inflammatory lipid metabolism of macrophages

doi: 10.1038/s41556-024-01457-0

Figure Lengend Snippet: a,b Western Blot analysis showing the knock-out efficiency of Pex5 −/− ( a ) and Abcd1 KD-efficiency ( b ) relative to control BMDMs representing n = 4;3 ( Pex5 −/− ; Abcd1 KD) biological repeats. c,d Images ( c ) and quantification ( d ) of M-P-LD clusters in LPS/IFN-γ-activated (24 h) BMDMs upon ABCD1 expression. Images represent maximum intensity projections. Scale bars: 5 µm, 2 µm (magnification). Boxes represent 25 th to 75 th percentiles, whiskers 10 th and 90 th percentiles. Dots are outliers, the median is shown as a line. Data represent N = 48;53 (BFP; ABCD1) cells from n = 3 biological replicates. P values were obtained using one-way ANOVA with Sidak’s post-hoc test. e-f PGE 2 levels of LPS/IFN-γ-treated (24 h) BMDMs expressing ABCD1 ( e ) or M1-Spastin ( f ) representing n = 3 biological replicates. P values were obtained using two-tailed, unpaired t tests ( e,f ). g,h Western Blot images ( g ) and quantification ( h ) showing protein levels of enzymes involved in FA release (ATGL, cPLA2) and PGE 2 biogenesis (COX2, mPGES1) in LPS/IFN-γ-activated BMDMs upon Abcd1 KD. Data represent n = 3 biologically independent experiments ( g,h ). P values were obtained using two-tailed, one-sample t tests ( h ). i-j Triglyceride analysis showing TG levels in LPS/IFN-γ-activated (24 h) Pex5 −/− ( i ) and Abcd1 KD BMDMs ( j ) relative to controls. Data represent n = 5;3 ( i,j ) biological replicates. P values were obtained using two-tailed, one-sample t tests. k,l TG arachidonic acid content ( k ) and release ( k,l ) of BFP and ABCD1 expressing cells from n = 3 biological replicates. P values were obtained using one-way ANOVA with Sidak’s post-hoc ( k ) and two-tailed, unpaired t tests ( l ) m Western Blot analysis showing the KO efficiency of Pex5 −/− Drp1 −/− and Drp1 −/− BMDMs obtained from LysM-Cre Drp1 fl/f Pex5 fl/fl and LysM-Cre Drp1 fl/fl mice, respectively. Data represent n = 3 biological replicates n Western Blot analysis showing the efficiency of Mfp2 and Gnpat KD relative to control BMDMs representative of n = 2 independent repeats. All bars show mean ± SEM ( e,f,h-l ). Numerical P values are available in Supplementary Information Table (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, not significant (ns) P > 0.05). Source numerical data and unprocessed blots are available in source data.

Article Snippet: The following antibodies were used for immunofluorescence staining (IF), western blot (WB) or flow cytometry (FACS): anti-ABCD1 (WB 1:5,000; IF 1:500; Abcam, ab197013), anti-ACSL1 (IF 1:200, Proteintech, 13989-1-AP), anti-ATGL (WB 1:1,000; IF 1:200; Cell Signaling Technology, 2439), anti-β-actin-horseradish peroxidase (HRP) (WB 1:40,000; SantaCruz, sc-47778 HRP), anti-Calnexin (IF 1:250; Proteintech, 10427-2-AP), anti-Catalase (IF 1:300; FACS 1:100; R&D Systems, AF3398), anti-CD16/CD23 (FACS 1:1,000; Thermo Fisher, 14-0161-82), anti-CD107a (IF 1:200; BioLegend, 121601), anti-CD107a Alexa488-conjugated (FACS 1:200; BioLegend, 121607), anti-COX2 (IF 1:400; WB 1:1,000; Cell Signaling Technology, 12282), anti-cPLA2 (IF 1:250; WB 1:1,000; Cell Signaling Technology, 5249), anti-DRP1 (WB 1:1,000, IF 1:100; Cell Signaling Technology, 8570), anti-phospho-DRP1 (S616) (WB 1:1,000; Cell Signaling Technology, 3455), anti-phospho-DRP1 (S637) (WB 1:1,000; Cell Signaling Technology, 4867), anti-FAM73B (WB 1:1,000; Abcam, ab122713), anti-GM130 (IF 1:300; FACS 1:500; BD Bioscience, 610823), anti-GNPAT (WB 1:1,000; Proteintech, 14931-1-AP), anti-HSD17B4 (WB 1:1,000; Novus Biologicals, NBP1-85296), anti-HSP60 (IF 1:1,000; FACS 1:500; Cell Signaling Technology, 12165), anti-HSP60 (IF 1:1,000, antibodies.com, A85438), anti-MFN2 (WB 1:1,000; Abcam, ab124773), anti-mPGES1 (IF 1:500; WB 1:1,000; Abcam, ab180589), anti-OPA1 (WB 1:1,000; Thermo Fisher, MA5-16149), anti-PEX5 (WB 1:5,000; Novus Biologicals, NBP1-87185), anti-RMDN3 (WB 1:200; Thermo Fisher, PA5-117028), anti-rabbit HRP (WB 1:8,000; ThermoFisher, 31460), anti-goat HRP (WB 1:10,000; ThermoFisher, 31402), anti-mouse HRP (WB 1:10,000; ThermoFisher, 61-6520), anti-rabbit biotin-conjugated (IF 1:500; Thermo Fisher, A16039), anti-rabbit Cy3 (IF 1:1,000; Jackson Immuno Research Laboratories, 111-165-144), anti-rabbit Alexa Fluor 568 (IF 1:500; Thermo Fisher, A10042), anti-rabbit Alexa Fluor 647 (IF 1:500, FACS 1:500; Thermo Fisher, A31573), anti-mouse DyLight 405 (IF 1:300; Jackson Immuno Research Laboratories, 715-475-151), anti-mouse Alexa Fluor 568 (IF 1:500, FACS 1:500; Thermo Fisher, A10037), anti-goat DyLight 405 (IF 1:300; Jackson Immuno Research Laboratories, 705-475-147), anti-goat Alexa 405 Plus (IF 1:500; Thermo Fisher, A48258), anti-goat Alexa Fluor 568 (IF 1:500; Thermo Fisher, A11057) anti-goat Alexa Fluor 647 (IF 1:500, FACS 1:500; Thermo Fisher, A32849), anti-rat DyLight 550 (IF 1:600; Thermo Fisher, SA5-10027) and anti-chicken Alexa Fluor 647 (IF 1:500, Jackson Immuno Research Laboratories, 703-605-155).

Techniques: Western Blot, Knock-Out, Control, Expressing, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Functional multi-organelle units control inflammatory lipid metabolism of macrophages

doi: 10.1038/s41556-024-01457-0

Figure Lengend Snippet: a , Images showing peroxisomes in wild-type or Pex5 −/− BMDMs representing n = 3 biological repeats. b – e , Images ( b , d ) and quantification ( c , e ) of M–ER–P–LD clusters in LPS/IFNγ-activated BMDMs upon Pex5 −/− ( b , c ) or Abcd1 knockdown (KD) ( d , e ) relative (rel.) to controls. shCtrl, scrambled shRNA control. Data from N = 42 (WT) and N = 44 ( Pex5 −/− ) and N = 31 (shCtrl) and N = 33 ( Abcd1 KD) cells were pooled from n = 3 biological replicates. P values were obtained using a one-way ANOVA with Sidak’s post hoc test ( c , e ). f , g , PGE 2 production of LPS/IFNγ-activated (24 h) Pex5 −/− ( f ) or Abcd1 KD ( g ) BMDMs relative to controls. Data represent n = 7 ( f ) and n = 5 ( g ) biologically independent experiments. P values were obtained using two-tailed, one-sample t -tests. h – j , Lipidomic analysis showing TG arachidonic acid content ( h , i ) and release ( h – j ) in LPS/IFNγ-treated Pex5 −/− ( h ), Abcd1 KD BMDMs ( i ) or Drp1 −/− and Pex5 −/− Drp1 −/− ( j ) BMDMs relative to controls. Data represent n = 5 ( h , vehicle) and n = 3 ( i , j and h , ATGLi) biological replicates. P values were obtained using one-way ANOVA with Sidak’s post hoc test ( h – j ). k , PGE 2 production of wild-type or Pex5 −/− BMDMs expressing DRP1 DN (LPS/IFNγ 24 h) from n = 4 biologically independent experiments. P values were obtained using a one-way ANOVA with Tukey’s post hoc test. l , m , Images ( l ) and quantification ( m ) of M–P–LD clusters LPS/IFNγ-activated DRP1 DN overexpressing BMDMs upon Abcd1 KD. Data represent N = 54 (shCtrl) and N = 54 ( Abcd1 KD) cells from n = 4 biological replicates. P values were obtained using Kruskal–Wallis with Dunn’s post hoc test. n , PGE 2 production of LPS/IFNγ-activated DRP1 DN overexpressing BMDMs upon Abcd1 , Gnpat or Mfp2 knockdown. Data represent n = 4 ( Abcd1 KD) or n = 3 ( Gnpat and Mfp2 KD) biological replicates. P values were obtained using two-tailed, one-sample t- tests. All images are maximum intensity projections. Scale bars, 5 µm and 2 µm (magnifications) ( a , b , d , l ). Box plots ( c , e , m ) are as in Fig. . Data are mean ± s.e.m. ( f – k , n ). Numerical P values are available in Supplementary Table . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source numerical data are available in .

Article Snippet: The following antibodies were used for immunofluorescence staining (IF), western blot (WB) or flow cytometry (FACS): anti-ABCD1 (WB 1:5,000; IF 1:500; Abcam, ab197013), anti-ACSL1 (IF 1:200, Proteintech, 13989-1-AP), anti-ATGL (WB 1:1,000; IF 1:200; Cell Signaling Technology, 2439), anti-β-actin-horseradish peroxidase (HRP) (WB 1:40,000; SantaCruz, sc-47778 HRP), anti-Calnexin (IF 1:250; Proteintech, 10427-2-AP), anti-Catalase (IF 1:300; FACS 1:100; R&D Systems, AF3398), anti-CD16/CD23 (FACS 1:1,000; Thermo Fisher, 14-0161-82), anti-CD107a (IF 1:200; BioLegend, 121601), anti-CD107a Alexa488-conjugated (FACS 1:200; BioLegend, 121607), anti-COX2 (IF 1:400; WB 1:1,000; Cell Signaling Technology, 12282), anti-cPLA2 (IF 1:250; WB 1:1,000; Cell Signaling Technology, 5249), anti-DRP1 (WB 1:1,000, IF 1:100; Cell Signaling Technology, 8570), anti-phospho-DRP1 (S616) (WB 1:1,000; Cell Signaling Technology, 3455), anti-phospho-DRP1 (S637) (WB 1:1,000; Cell Signaling Technology, 4867), anti-FAM73B (WB 1:1,000; Abcam, ab122713), anti-GM130 (IF 1:300; FACS 1:500; BD Bioscience, 610823), anti-GNPAT (WB 1:1,000; Proteintech, 14931-1-AP), anti-HSD17B4 (WB 1:1,000; Novus Biologicals, NBP1-85296), anti-HSP60 (IF 1:1,000; FACS 1:500; Cell Signaling Technology, 12165), anti-HSP60 (IF 1:1,000, antibodies.com, A85438), anti-MFN2 (WB 1:1,000; Abcam, ab124773), anti-mPGES1 (IF 1:500; WB 1:1,000; Abcam, ab180589), anti-OPA1 (WB 1:1,000; Thermo Fisher, MA5-16149), anti-PEX5 (WB 1:5,000; Novus Biologicals, NBP1-87185), anti-RMDN3 (WB 1:200; Thermo Fisher, PA5-117028), anti-rabbit HRP (WB 1:8,000; ThermoFisher, 31460), anti-goat HRP (WB 1:10,000; ThermoFisher, 31402), anti-mouse HRP (WB 1:10,000; ThermoFisher, 61-6520), anti-rabbit biotin-conjugated (IF 1:500; Thermo Fisher, A16039), anti-rabbit Cy3 (IF 1:1,000; Jackson Immuno Research Laboratories, 111-165-144), anti-rabbit Alexa Fluor 568 (IF 1:500; Thermo Fisher, A10042), anti-rabbit Alexa Fluor 647 (IF 1:500, FACS 1:500; Thermo Fisher, A31573), anti-mouse DyLight 405 (IF 1:300; Jackson Immuno Research Laboratories, 715-475-151), anti-mouse Alexa Fluor 568 (IF 1:500, FACS 1:500; Thermo Fisher, A10037), anti-goat DyLight 405 (IF 1:300; Jackson Immuno Research Laboratories, 705-475-147), anti-goat Alexa 405 Plus (IF 1:500; Thermo Fisher, A48258), anti-goat Alexa Fluor 568 (IF 1:500; Thermo Fisher, A11057) anti-goat Alexa Fluor 647 (IF 1:500, FACS 1:500; Thermo Fisher, A32849), anti-rat DyLight 550 (IF 1:600; Thermo Fisher, SA5-10027) and anti-chicken Alexa Fluor 647 (IF 1:500, Jackson Immuno Research Laboratories, 703-605-155).

Techniques: Knockdown, shRNA, Control, Two Tailed Test, Expressing

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Illustration depicting the acute corticosterone treatment in the dorsal hippocampus. B Representative dorsal hippocampal slices immunolabeled with antibody against TRIP8b. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic TRIP8b protein expression. C Quantification of TRIP8b protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. D Representative dorsal hippocampal slices immunolabeled with antibody against HCN1. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 protein expression. E Quantification of HCN1 protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. F , G We performed cell-attached voltage-clamp recordings. F Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). G I h was significantly elevated in the dorsal CA1 neurons from corticosterone treatment compared with those from the vehicle-treated group. Dexamethasone reduced R in at RMP ( H ) and at −65 mV ( J ) in dorsal CA1 neurons. Corticosterone-induced decrease in R in at RMP ( H ) and at −65 mV ( J ) was blocked by RU 486, KT5720, and ZD7288. Changes in R in at RMP ( I ) and at −65 mV ( K ) were blocked by RU 486, KT5720, and ZD7288. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Immunolabeling, Expressing

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Timeline of CSDS, behavioral tests, electrophysiology, and biochemical assay. B CSDS produced the susceptible and resilient phenotype during the social interaction test. After 1 month ( C ) or 3 months ( D ) of no CSDS, susceptible mice showed persistent social avoidance during the social interaction test. E , F We performed whole-cell current-clamp recordings. E Representative voltage responses with depolarizing current step (210 pA; 750 ms) at RMP in dorsal CA1 neurons. F Dorsal CA1 neurons of susceptible group had lower action potential firing than control group, whereas the resilient group had higher action potential firing. G , H We performed cell-attached voltage-clamp recordings. G Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). H I h was significantly elevated in the dorsal CA1 neurons from susceptible group compared with those from the control and resilient mice. Representative dorsal hippocampal slices immunolabeled with antibody against HCN1 ( I ) and TRIP8b ( K ). Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 and TRIP8b protein expression. Quantification of HCN1 ( J ) and TRIP8b ( L ) protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. M , N We performed whole-cell current-clamp recordings. M Corticosterone reduced R in at RMP of the dorsal CA1 neurons in the control and resilient groups, but had no effect on R in in susceptible group. N Changes in R in at RMP were much lower in susceptible group compared to the control and resilient groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Produced, Control, Immunolabeling, Expressing

Journal: Experimental & Molecular Medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: a HepG2 cells were treated with CTL siRNA (100 nM) and PEX5 siRNA #1 or #2 (100 nM) for 24 h. Then, cells were incubated in the presence or absence of serum. After 24 h, cells were harvested and immunoblotted with antibodies against PEX5, LC3I/II, p62, and β-actin. b Immunoblot analysis of p-70S6K, p-S6R, and p-4E-BP-1, and total 70S6K, S6R, and 4E-BP-1. c Cells were transfected with GFP-LC3 plasmid. After 6 h of transfection, cells were treated with CTL and PEX5 siRNAs. Visualization and quantification of GFP-LC3 puncta were performed by fluorescence microscopy after 48 h of incubation. Representative images from three independent determinations are shown (* P < 0.05). Scale bar: 10 μm. d Immunoblot analysis of TSC2 and PEX5 protein levels. e Transcript levels of TSC2 were measured by Q-PCR (** P < 0.01)

Article Snippet: They were then transfected with 100 nM control siRNA, PEX5 siRNA (#1 sense-CGUAUCCUGGGAUCUCUCU, antisense-AGAGAGAUCCCAGGAUACG, #2 sense-GAAUUCAUCUCUGAAGUUA, antisense-UAACUUCAGAGAGAUGAAUUC-3, Bioneer, Daejeon, Korea), or TFEB siRNA (sc38509, Santa Cruz Biotechnology) for 24 h in serum-free medium, using Lipofectamine RNAiMax transfection reagent (Invitrogen/Life Technologies), and then exposed to serum starvation for 24 h. The effect of gene silencing was determined by Q-PCR and western blot analysis.

Techniques: Incubation, Western Blot, Transfection, Plasmid Preparation, Fluorescence, Microscopy

Journal: Experimental & Molecular Medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: a Cells were treated with CTL and PEX5 siRNAs (100 nM) for 24 h. Then, cells were incubated in the presence or absence of serum. After 24 h, cells were stained with an antibody against TFEB and analyzed by fluorescence microscopy. Scale bar: 10 μm. b After HepG2 cells were treated as indicated, cells were subjected to nuclear and cytosol fractionation and immunoblotted with antibodies against TFEB, PCNA, and β-actin. PCNA and β-actin were used as nuclear and cytosolic markers, respectively. c , d Cells were transfected with CTL or TFEB siRNA (100 nM) for 24 h, and then incubated in the presence or absence of serum for 24 h. Protein expression of TFEB, LC3 and SQSTM1/p62 (autophagy proteins), and PMP70, DBP, and catalase (peroxisomal proteins) were measured by immunoblotting. e Q-PCR analysis of peroxisomal genes was performed (** P < 0.01, * P < 0.05)

Article Snippet: They were then transfected with 100 nM control siRNA, PEX5 siRNA (#1 sense-CGUAUCCUGGGAUCUCUCU, antisense-AGAGAGAUCCCAGGAUACG, #2 sense-GAAUUCAUCUCUGAAGUUA, antisense-UAACUUCAGAGAGAUGAAUUC-3, Bioneer, Daejeon, Korea), or TFEB siRNA (sc38509, Santa Cruz Biotechnology) for 24 h in serum-free medium, using Lipofectamine RNAiMax transfection reagent (Invitrogen/Life Technologies), and then exposed to serum starvation for 24 h. The effect of gene silencing was determined by Q-PCR and western blot analysis.

Techniques: Incubation, Staining, Fluorescence, Microscopy, Fractionation, Transfection, Expressing, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: Cells were transfected with CTL siRNA (100 nM) and PEX5 siRNA (100 nM) for 24 h, and then incubated in the presence or absence of serum for 24 h. After incubation, cells were stained with LysoTracker Red DND-99 (50 nM) for 3 h and analyzed by confocal microscopy ( a ) or fluorescence-activated cell sorting and quantified ( b ). Data are presented as mean ± s.d. from three independent experiments (* P < 0.05). c Transcript levels of TFEB target genes were measured by Q-PCR. Values are expressed as fold increase compared to the starvation control group. Data were presented as mean ± s.d. from three independent experiments (** P < 0.01, * P < 0.05). d , e Protein expression of LAMP-1/LAMP-2 (lysosomal proteins) and cathepsins B/D (lysosomal proteases) were measured by immunoblotting

Article Snippet: They were then transfected with 100 nM control siRNA, PEX5 siRNA (#1 sense-CGUAUCCUGGGAUCUCUCU, antisense-AGAGAGAUCCCAGGAUACG, #2 sense-GAAUUCAUCUCUGAAGUUA, antisense-UAACUUCAGAGAGAUGAAUUC-3, Bioneer, Daejeon, Korea), or TFEB siRNA (sc38509, Santa Cruz Biotechnology) for 24 h in serum-free medium, using Lipofectamine RNAiMax transfection reagent (Invitrogen/Life Technologies), and then exposed to serum starvation for 24 h. The effect of gene silencing was determined by Q-PCR and western blot analysis.

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, Fluorescence, FACS, Control, Expressing, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: Cells transfected with CTL siRNA (100 nM) and PEX5 siRNA (100 nM) for 24 h were pretreated with DMSO or rapamycin (1 μM) for 6 h, and then incubated in the absence of serum for 24 h. ( a ) After HepG2 cells were treated as indicated, cells were stained with an antibody against TFEB and analyzed by fluorescence microscopy. Scale bar: 10 μm. Data are presented as mean ± SD from three independent experiments (** P < 0.01). ( b ) After HepG2 cells were treated as indicated, cells were subjected to nuclear and cytosol fractionation and immunoblotted with antibodies against TFEB, PCNA, and β-actin. PCNA and β-actin were used as nuclear and cytosolic markers, respectively. ( c ) Immunoblot analysis of LC3 and SQSTM1/p62. ( d ) Immunoblot analysis of PEX5, p-70S6K, p-S6R, and p-4E-BP-1

Article Snippet: They were then transfected with 100 nM control siRNA, PEX5 siRNA (#1 sense-CGUAUCCUGGGAUCUCUCU, antisense-AGAGAGAUCCCAGGAUACG, #2 sense-GAAUUCAUCUCUGAAGUUA, antisense-UAACUUCAGAGAGAUGAAUUC-3, Bioneer, Daejeon, Korea), or TFEB siRNA (sc38509, Santa Cruz Biotechnology) for 24 h in serum-free medium, using Lipofectamine RNAiMax transfection reagent (Invitrogen/Life Technologies), and then exposed to serum starvation for 24 h. The effect of gene silencing was determined by Q-PCR and western blot analysis.

Techniques: Transfection, Incubation, Staining, Fluorescence, Microscopy, Fractionation, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: Cells transfected with siRNA for 24 h were pretreated with DMSO or rapamycin (1 μM) for 6 h, and then incubated in the absence of serum for 24 h. a Transcript levels of peroxisome-related genes were measured by Q-PCR. Values are expressed as fold increase compared to the starvation control group. Data are presented as mean ± s.d. from three independent experiments (** P < 0.01, * P < 0.05). b Immunoblot analysis of PMP70, ACOX1, DBP, catalase, and PEX5. c Q-PCR analysis of TFEB-regulated genes. Data are presented as mean ± s.d. from three independent experiments (** P < 0.01, * P < 0.05). d Protein expression of LAMP-1/LAMP-2 (lysosomal proteins) and cathepsins B/D (lysosomal proteases) were measured by immunoblotting. e Cells were transfected with empty, GFP-WT-TFEB, and GFP-TFEB-S142A plasmids. After 6 h of transfection, cells were treated with CTL and PEX5 siRNAs. After incubation in the absence of serum for 24 h, cells were subjected to immunoblot analysis with antibodies against PEX5, LC3, LAMP1, PMP70, TFEB, and β-actin

Article Snippet: They were then transfected with 100 nM control siRNA, PEX5 siRNA (#1 sense-CGUAUCCUGGGAUCUCUCU, antisense-AGAGAGAUCCCAGGAUACG, #2 sense-GAAUUCAUCUCUGAAGUUA, antisense-UAACUUCAGAGAGAUGAAUUC-3, Bioneer, Daejeon, Korea), or TFEB siRNA (sc38509, Santa Cruz Biotechnology) for 24 h in serum-free medium, using Lipofectamine RNAiMax transfection reagent (Invitrogen/Life Technologies), and then exposed to serum starvation for 24 h. The effect of gene silencing was determined by Q-PCR and western blot analysis.

Techniques: Transfection, Incubation, Control, Western Blot, Expressing

Journal: iScience

Article Title: Alterations in ether phospholipids metabolism activate the conserved UPR-Xbp1- PDIA3/ERp60 signaling to maintain intestinal homeostasis

doi: 10.1016/j.isci.2025.111946

Figure Lengend Snippet:

Article Snippet: The Pex5-loxP mouse (B6J.129- Pex5 tm1Pec /BaesJ, Jackson Laboratory) contains loxP sites flanking exons 11–14 of the Pex5 gene, and deletion of the floxed sequence creates a null allele.

Techniques: Recombinant, Qubit Protein Assay, In Situ, Gentle, Software, Microscopy