Journal: Nature

Article Title: Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway

doi: 10.1038/s41586-020-2078-2

Figure Lengend Snippet: (a) Schematic of screen. (b) Comparison of gene scores (defined in Methods) from the screen in untreated and oligomycin-treated conditions. (c) Knockdown of DELE1 quantified by qPCR ( n = 3 technical replicates). (d) Reporter activation in cells expressing a non-targeting control sgRNA (NTC) or DELE1 sgRNAs (mean ± s.d., n = 3 culture wells). (e) Immunoblot of endogenous ATF4. Cells were treated with 1.25 ng/mL of oligomycin for 16 hrs (Oli) or untreated (Un). Left , representative blot; Right , quantification of n = 2 blots. (f) ATF4 reporter activation in cells transiently overexpressing (OE) DELE1-mClover and expressing sgRNA to knockdown (KD) HRI where indicated. (mean ± s.d., n = 3 culture wells). (g) ATF4 reporter activation in cells transiently overexpressing (OE) HRI and expressing sgRNA to knockdown (KD) DELE1 where indicated. (mean ± s.d., n = 3 culture wells). (h) DELE1-mClover expression from the AAVS1 safe-harbor locus is not sufficient to induce the ATF4 reporter in the absence of stress, but complements DELE1 knockdown upon oligomycin treatment (mean ± s.d., n = 3 culture wells). (i) Co-localization of DELE1-mClover with the mitochondrial-targeted mRuby (Mito7-mRuby). Scale bar, 7 μm. Similar results for n = 2 culture wells.

Article Snippet: HEK293T cells and derived cell lines were seeded in the 8-well chamber slide (Ibidi #80824) for 24 h and transfected with DELE1 truncated constructs and/or plasmid mito7-mRuby (a gift from Michael Davidson, Addgene plasmid # 55874) as indicated, using TransIT-Lenti Transfection Reagent (Mirus #MIR 6600).

Techniques: Comparison, Knockdown, Activation Assay, Expressing, Control, Western Blot

Journal: Nature

Article Title: Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway

doi: 10.1038/s41586-020-2078-2

Figure Lengend Snippet: ( a-b ) Zoom-out views for two-color 3D-STORM super-resolution images of DELE1-mClover vs. TOM20 and Hsp60. Two-color DELE1-mClover (magenta) vs. (a) TOM20 (green) or (b) Hsp60 (green), followed by the two separated color channels. Scale bars: 1 μm. The boxed regions correspond to , . Similar results were obtained in n = 3 independent experiments. ( c ) Biochemical fractionation indicates that DELE1 associates with mitochondrial membranes. Cells stably expressing DELE1-mClover were fractionated into cytosol and mitochondria. Mitochondria were incubated in either isotonic buffer (10 mM Tris HCl, pH 6.7, 0.15 mM MgCl 2 0.25mM sucrose,1 mM DTT, protease inhibitor cocktail (Sigma #5892970001)) or H 2 O (extreme hypotonic condition) for 5 min, followed by centrifugation (10000 xg for 10 min) to separate the supernatant and pellet. The pellet was either dissolved with RIPA buffer or incubated with 0.1M NaCO 3 (pH=11.4) for 30 min at 4°C. Supernatant and pellet from NaCO 3 -treated samples were collected for WB. Unlike soluble matrix protein LonP1 and HSPD1, which can be extracted with H 2 O incubation, only small proportion of DELE1 is present in the supernatant. NaCO 3 can extract the majority of the DELE1 S but not DELE1 L , which is similar to the pattern of mitochondrial membrane protein VDAC, suggesting that DELE1 L is more likely a membrane associated protein. n = 2 independent experiments. For gel source data, see . ( d ) The indicated DELE1-mClover constructs were transiently overexpressed in reporter cells, and reporter induction was quantified by flow cytometry (mean ± s.d., n = 3 culture wells). Subcellular localization was evaluated by microscopy in cells also expressing mitochondrial-targeted mRuby. ( e ) Lack of co-localization of transiently expressed DELE1ΔN100-mClover and DELE1ΔN148-mClover (green) with the mitochondrial stain Mitotracker (red). Scale bar, 7 μm. Similar results obtained in n = 2 culture wells. ( f ) Increased detection of DELE1-mClover outside the mitochondria upon oligomycin treatment. 3D-STORM super-resolution images of stably expressed DELE1-mClover (colors indicating depth in the z dimension) in untreated cells ( left ) and cells treated with 1.25 ng/mL oligomycin for 16 h ( right ). Areas boxed in red in the top panels are shown in higher magnification in the bottom panels. Similar results were obtained in n = 3 independent experiments. ( g ) Co-localization of transiently expressed HRI-mClover and DELE1-mClover with the mitochondrial-targeted mRuby (Mito7-mRuby). Scale bar, 7 μm. n = 1 culture well.

Article Snippet: HEK293T cells and derived cell lines were seeded in the 8-well chamber slide (Ibidi #80824) for 24 h and transfected with DELE1 truncated constructs and/or plasmid mito7-mRuby (a gift from Michael Davidson, Addgene plasmid # 55874) as indicated, using TransIT-Lenti Transfection Reagent (Mirus #MIR 6600).

Techniques: Fractionation, Stable Transfection, Expressing, Incubation, Protease Inhibitor, Centrifugation, Membrane, Construct, Flow Cytometry, Microscopy, Staining

Journal: International Journal of Biological Sciences

Article Title: SEC23A Inhibit Melanoma Metastatic through Secretory PF4 Cooperation with SPARC to Inhibit MAPK Signaling Pathway

doi: 10.7150/ijbs.60866

Figure Lengend Snippet: Sec23a silencing significantly enhances the metastatic behavior of melanoma cells in vitro . (A, B) , RT-qPCR and western blot were performed to confirm effective Sec23a silencing; (C) Quantitative analysis of the expressions of SEC23A in OL-N.C.,OL-shSec23a,POL-Vetor and POL-Sec23a-OE. (D,F) Sec23a knockdown significantly enhanced migration and invasion; (E,G) Overexpression of Sec23a inhibits invasion and migration of melanoma cells; (H,I) Effect of altered Sec23a expression on colony formation; (J) Cell proliferation was not affected by Sec23a expression changes, measured by CCK8 assay. (*p<.05,**p<.01,***p<.001).

Article Snippet: Sec23a overexpression (OE) plasmid (Plasmid #36158) and empty vector were obtained from Addgene.

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Knockdown, Migration, Over Expression, Expressing, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: SEC23A Inhibit Melanoma Metastatic through Secretory PF4 Cooperation with SPARC to Inhibit MAPK Signaling Pathway

doi: 10.7150/ijbs.60866

Figure Lengend Snippet: Mass spectrometry (MS) detection of differentially secreted proteins in OL-N.C. and OL-shSec23a supernatant. (A) quantification of differentially down-regulated secretory proteins; (B) STRING database analysis derived protein-protein interaction (PPI) network of the 28 down-regulated secretory proteins; (C) correlation of 28 down- regulated secretory proteins and Sec23a from TCGA analysis of melanoma datasets; (D) expression of Pf4 and Ppbp between melanoma cells and melanocyte in GSE22301; (E) expression of Pf4 and Ppbp between low- and high-metastatic melanoma cells in GSE114380 ; (F) expression of Pf4 and Ppbp between in OL vs POL cells; (G) expression of Pf4 in OL vs OL-shSec23a; (H) validation of the Pf4 down-regulation in OL-shSec23a and POL cells using conditioned medium, respectively. (I) Quantitative analysis of the expressions of Pf4 in the supernatants of OL-N.C.,OL-shSec23a,OL and POL cells (*p<.05,**p<.01,***p<.001).

Article Snippet: Sec23a overexpression (OE) plasmid (Plasmid #36158) and empty vector were obtained from Addgene.

Techniques: Mass Spectrometry, Derivative Assay, Expressing, Biomarker Discovery

Journal: International Journal of Biological Sciences

Article Title: SEC23A Inhibit Melanoma Metastatic through Secretory PF4 Cooperation with SPARC to Inhibit MAPK Signaling Pathway

doi: 10.7150/ijbs.60866

Figure Lengend Snippet: Pf4 silencing in OL and POL-Sec23a-OE cells correlates with the invasive behavior of melanoma cells. (A) Real-time PCR validation of Pf4 silencing; (B) western blot analysis of PF4 secretion in conditioned media upon Pf4 silencing; (C) Quantitative analysis of the expressions of PF4 in the supernatants of OL-N.C. ,OL-shPf4,POL-Sec23a-shNC and POL-Sec23a-shPf4. (D-G) Pf4 silencing significantly enhanced melanoma cell migration and invasion ( Material and Methods ); (H, I) Pf4 silencing significantly enhanced colony formation of melanoma cells; (J) Pf4 silencing did not alter cell proliferation, measured by CCK8 assay (*p<.05,**p<.01,***p<.001).

Article Snippet: Sec23a overexpression (OE) plasmid (Plasmid #36158) and empty vector were obtained from Addgene.

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Western Blot, Migration, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: SEC23A Inhibit Melanoma Metastatic through Secretory PF4 Cooperation with SPARC to Inhibit MAPK Signaling Pathway

doi: 10.7150/ijbs.60866

Figure Lengend Snippet: Alteration of secreted PF4 affects the invasive behavior of melanoma cells. Neutralization of PF4 with PF4 antibody (10ug/ml) reversed the inhibitory effect of Pf4 on OL ( A, C ) and POL-Sec23a-OE ( B, D ) cell migration and invasion; Recombinant PF4 protein (450 ng/ml) treatment inhibited POL cell ( E, G ); and augmented OL-shSec23a cell (F, H) migration and invasion . (I, J) Recombinant PF4 protein (450 ng/ml) treatment prevented Pf4 down regulation-induced increase in colony formation in POL and OL-shSec23a cells (*p<.05, **p<.01,***p<.001).

Article Snippet: Sec23a overexpression (OE) plasmid (Plasmid #36158) and empty vector were obtained from Addgene.

Techniques: Neutralization, Migration, Recombinant

Journal: International Journal of Biological Sciences

Article Title: SEC23A Inhibit Melanoma Metastatic through Secretory PF4 Cooperation with SPARC to Inhibit MAPK Signaling Pathway

doi: 10.7150/ijbs.60866

Figure Lengend Snippet: Stable Pf4 silencing significantly enhances the metastatic capability of melanoma cells in NOD/SCID mice. (A, E) Photographic representation of macroscopic metastases of NOD/SCID mice 4 weeks after tail vein injection of control and Pf4 knockdown cells (POL-Sec23a-OE-shPf4 and OL-shPf4) (Four mice per group); (B, F) Quantitative analysis of the surface metastatic foci in control and Pf4 knockdown cells; (C,G) representative whole-lung images to visualize metastases on the lung (asterisk) by H&E staining; (D,H) body weight changes of mice receiving control and Pf4 knockdown cells (*p<.05,**p<.01,***p<.001).

Article Snippet: Sec23a overexpression (OE) plasmid (Plasmid #36158) and empty vector were obtained from Addgene.

Techniques: Injection, Control, Knockdown, Staining

Journal: International Journal of Biological Sciences

Article Title: SEC23A Inhibit Melanoma Metastatic through Secretory PF4 Cooperation with SPARC to Inhibit MAPK Signaling Pathway

doi: 10.7150/ijbs.60866

Figure Lengend Snippet: Pf4 acts downstream of Sec23a to inhibit MAPK/ERK activation. (A) KEGG pathway enrichment analysis for Pf4-related genes in GSE22301 and GSE46517 and prioritized MAPK/ERK pathway; (B) (left) Pf4 knockdown augmented ERK/MAPK activation in OL and POL-Sec23a-OE cells; (right) Quantitative analysis of the expressions of p-ERK in OL-N.C. ,OL-shPf4,POL-Sec23a-shNC and POL-Sec23a-shPf4 (C) (top) recombinant PF4 protein (450 ng/ml) treatment inhibited ERK/MAPK activation in OL-shSec23a and POL cell; (bottom) Quantitative analysis of the expressions of p-ERK in PBS and rPF4. (D) (left) PD98059 (20uM) treatment prevented the increase of p-ERK caused by Pf4 knockdown in OL-shPf4; (right) Quantitative analysis of the expressions of p-ERK in DMSO and PD98059. (E) Pf4 knockdown had no significant effect on AKT activation in OL cells; (F-I) PD98059 (20uM) treatment prevented the increase of OL cell invasion and migration caused by Pf4 knockdown (OL-shPf4 cells); (J,K) PD98059(20uM) treatment prevented the increase of colony formation in POL-Sec23a and OL cells caused by Pf4 knockdown (OL-shPf4 and POL-Sec23a-OE-shPf4 cells). ( *p<.05, **p<.01,***p<.001).

Article Snippet: Sec23a overexpression (OE) plasmid (Plasmid #36158) and empty vector were obtained from Addgene.

Techniques: Activation Assay, Knockdown, Recombinant, Migration

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: MAP4 projection domains restrict its association to tyrosinated microtubules. (a) The Schematic of MAP4 truncation constructs used in the study. (b-e) Representative confocal images of BS-C-1 cells transiently expressing GFP-tagged MAP4 constructs (magenta) immunostained for detyrosinated microtubules (green) along with corresponding insets show magnified views of the regions indicated by yellow boxes. (f) Box plot quantifying MAP4 colocalization with detyrosinated microtubules. (g) Model illustrating the role of MAP4 projection domains in conferring specificity for tyrosinated microtubules. Data represents mean (line) ± SD (box). Number of cells (n) analyzed for MAP4-FL (10), MAP4-MTBD (11), MAP4-ΔN (15) and MAP4-ΔC (14). Statistical significance was assessed using the Mann– Whitney U test (*p<0.05, **p<0.01, not significant [ns]). Scale bars: 10 μm.

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques: Construct, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: Kinesin-1 and kinesin-3 rigor mutants differentially associate with MAP4 and MAP7D1 (a-b) Confocal images of BS-C-1 cells transiently expressing GFP-tagged (a) KIF5B-R (green) or (b) KIF1A-R (green), immunostained for endogenous MAP4 (magenta). (c) Bar plot of Mander’s colocalization quantification shows significantly higher colocalization of MAP4 with KIF1A-R ( n = 8 cells) than KIF5B-R ( n = 9 cells). (d-e) Confocal images of cells expressing GFP-tagged (d) KIF1A-R or (e) KIF5B-R (green), immunostained for endogenous MAP7D1 (magenta). (f) Bar plot of Mander’s colocalization quantification reveals significantly higher colocalization of MAP7D1 with KIF5B-R ( n = 10 cells) than KIF1A-R ( n = 9 cells). (g–h) Co-expression of KIF5B-R (green) with (g) MAP4-FL or (h) MAP4-MTBD (magenta), Line intensity profiles show MAP4-FL is excluded from KIF5B-R-decorated microtubules, while MAP4-MTBD exhibits extensive colocalization. (i) Model illustrating the preferential association of KIFB-R and KIF1A-R with MAP7D1 and MAP4, respectively. Bars represent the mean; whiskers indicate standard deviation from three independent experiments. Statistical significance was assessed using the Mann–Whitney U test (****p < 0.0001). Scale bars: 10 μm.

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques: Expressing, Standard Deviation, MANN-WHITNEY