rab7a expression plasmid  (OriGene)

Journal: medRxiv

Article Title: Biallelic WDR91 variants cause a neurodevelopmental disorder through impaired endosomal maturation and autophagy dysregulation

doi: 10.64898/2026.04.03.26349989

Figure Lengend Snippet: A. Time-lapse recording of dynamic changes of the early endosomal marker 2×FYVE-mCherry and the late endosomal protein GFP-Rab7 on endosomes in transduced HEK193 cells (left) and duration of the overlap of 2×FYVE and Rab7 on endosomes (right; one-way ANOVA with Kruskal-Wallis multiple comparison test, ** p<0.01, **** p<10 - ; at least 10 endosomes per cell line were analyzed). B. Colocalization of endogenous EEA1 with Rab7 in WDR91 WT and WDR91 Y15N cells: representative confocal images (left) and assessment of Pearson’s coefficient (right; Mann-Whitney test, *** p<10 - ; at least 30 independent cells per cell line were analyzed). C. EEA1 and Rab7 protein expression in transduced HEK293 cells, representative blot of three independent experiments. Individual fluorescence channels were analyzed separately due to distinct background signal. D. Colocalization of WDR91 with endogenous EEA1 or Rab7 in WDR91 WT and WDR91 Y15N cells: representative confocal images (left) and assessment of Pearson’s coefficient (right; 2-way ANOVA with Sidak multiple comparison test, *** p<10 -3 , **** p<10 -4 ; at least 30 independent cells per cell line were analyzed).

Article Snippet: Cells were then saturated using TBS-Tween 0.01% supplemented with 3% goat serum, and stained with a primary anti-human WDR91 Ab (Abcam) and anti-human EEA1 or anti-human Rab7 Abs (Cell Signaling Technologies) and a secondary goat anti-mouse/rabbit IgG mAb coupled to AF488 or AF687 (Thermo Fisher Scientific).

Techniques: Marker, Comparison, MANN-WHITNEY, Expressing, Fluorescence

Journal: Advanced Science

Article Title: Extracellular Vesicle‐Delivered tRF‐His‐GTG‐1 Reprograms Neutrophil Lipophagy and Triggers Inflammation in COVID‐19

doi: 10.1002/advs.202508695

Figure Lengend Snippet: Spike protein and pEV spike impair lipophagy in neutrophils via TLR8–mTOR activation. (A) TEM images showing markedly increased lipid droplets (LDs; red arrows) in neutrophils from patients with COVID‐19 and in spike protein‐treated healthy neutrophils, with LDs frequently adjacent to lysosomes (Ly; blue arrows), indicative of impaired lipophagic processing. Scale bar: 500 nm. (B) Immunoblot analysis of neutrophils from patients with moderate/severe COVID‐19, long COVID, recovered patients, and SARS‐CoV‐2‐infected hamsters showing increased expression of TLR8, total and phosphorylated mTOR (Ser2448), phosphorylated S6K (Thr389), LC3‐II, p62, and PLIN2, together with reduced RAB7A, consistent with impaired lipophagy. (C) Spike protein or pEV spike alone induced similar autophagy defects, whereas cotreatment synergistically enhanced TLR8 activation, mTOR signaling, LC3‐II/p62 accumulation, and RAB7A suppression. These effects were largely reversed by the TLR8‐specific inhibitor Cu‐CPT9a. (D) Immunofluorescence analysis showing LD accumulation and reduced LD–lysosome (LAMP1⁺) colocalization in spike‐treated neutrophils, further exacerbated by pEV spike co‐treatment. Rapamycin restored LD–lysosome fusion, as reflected by increased yellow puncta. Scale bar: 5 µm. (E) Immunoblotting revealed that rapamycin reversed spike and pEV spike ‐induced increases in mTOR activation, LC3‐II/p62 accumulation, but had no effect on RAB7A expression. All experiments were performed in triplicate. The data are presented as mean ± SD. Statistical significance was assessed using the Kruskal–Wallis test with Dunn's post hoc correction for multiple groups, and the Mann–Whitney U ‐test or unpaired two‐tailed Student's t ‐test for two‐group comparisons, as described in the Experimental Section. *** p < 0.005. Densitometric analysis of immunoblotting is provided in Supporting Information . Abbreviations: HC, healthy controls; LC, patients with long COVID; M/SC, patients with moderate/severe COVID‐19; RC, patients who had recovered from COVID‐19.

Article Snippet: Human primary neutrophils (2 × 106 cells per reaction) were transiently transfected with 500 ng DNA of the RAB7A expression plasmid (OriGene, RC201776) or an empty control vector using the Neon Transfection System Kit (Thermo Fisher Scientific, MPK1025) according to the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Infection, Expressing, Immunofluorescence, MANN-WHITNEY, Two Tailed Test

Journal: Advanced Science

Article Title: Extracellular Vesicle‐Delivered tRF‐His‐GTG‐1 Reprograms Neutrophil Lipophagy and Triggers Inflammation in COVID‐19

doi: 10.1002/advs.202508695

Figure Lengend Snippet: Spike protein and pEV spike suppress RAB7A transcription in neutrophils through tRF‐His‐GTG‐1 targeting. (A) An immunofluorescence assay showed increased colocalization of LDs with lysosomes (LAMP1⁺) in RAB7A‐overexpressing neutrophils, indicating restored lipophagy. (B) Immunoblotting showed that RAB7A overexpression attenuated spike‐ and pEV spike ‐induced PLIN2 upregulation, supporting a role for RAB7A in limiting LD accumulation. (C) RNA‐seq analysis revealed that spike protein suppressed RAB7A mRNA expression in neutrophils. (D) Heatmap of autophagy‐ and lysosome‐related gene expression in human neutrophils treated with SARS‐CoV‐2 spike protein in the absence or presence of a tRF‐His‐GTG‐1 inhibitor. (E,F) qRT‐PCR analysis confirmed reduced RAB7A expression in neutrophils from (E) patients with COVID‐19 and (F) SARS‐CoV‐2‐infected hamsters, relative to HC. (G) Spike protein and pEV spike synergistically suppressed RAB7A transcription in human neutrophils. (H) Bioinformatic analysis predicted a tRF‐His‐GTG‐1 binding site in the 3′ UTR of RAB7A mRNA. (I) Transfection of a tRF‐His‐GTG‐1 mimic into human neutrophils reduced endogenous RAB7A mRNA expression. (J) Luciferase reporter assays confirmed that tRF‐His‐GTG‐1 directly targets the RAB7A 3′ UTR; mutation of the predicted binding site (MT) abolished this suppressive effect. Scale bars in immunofluorescence images represent 5 µm. All experiments were performed in triplicate. The data are presented as mean ± SD. Statistical significance was assessed using the Kruskal–Wallis test with Dunn's post hoc correction for multiple groups, and the Mann–Whitney U ‐test or unpaired two‐tailed Student's t ‐test for two‐group comparisons, as described in the Experimental Section. ** p < 0.01, *** p < 0.005; ns, not significant. Densitometric analysis of immunoblotting is provided in Supporting Information . Abbreviations: HC, healthy controls; LC, patients with long COVID; M/SC, patients with moderate/severe COVID‐19; MT, mutant; RC, patients who had recovered from COVID‐19; WT, wild type.

Article Snippet: Human primary neutrophils (2 × 106 cells per reaction) were transiently transfected with 500 ng DNA of the RAB7A expression plasmid (OriGene, RC201776) or an empty control vector using the Neon Transfection System Kit (Thermo Fisher Scientific, MPK1025) according to the manufacturer's instructions.

Techniques: Immunofluorescence, Western Blot, Over Expression, RNA Sequencing, Expressing, Gene Expression, Quantitative RT-PCR, Infection, Binding Assay, Transfection, Luciferase, Mutagenesis, MANN-WHITNEY, Two Tailed Test

Journal: Advanced Science

Article Title: Extracellular Vesicle‐Delivered tRF‐His‐GTG‐1 Reprograms Neutrophil Lipophagy and Triggers Inflammation in COVID‐19

doi: 10.1002/advs.202508695

Figure Lengend Snippet: COVID‐19‐associated pEVs carry abundant tRF‐His‐GTG‐1 that impairs lipophagy in neutrophils via TLR8–mTOR activation and RAB7A suppression. (A,B) qRT‐PCR analysis showed elevated tRF‐His‐GTG‐1 levels in pEVs derived from (A) recombinant spike protein‐treated human platelets and (B) SARS‐CoV‐2‐infected hamsters. (C) tRF‐His‐GTG‐1 levels in pEVs from patients with acute COVID‐19 (with different disease severities and clinical outcomes) and HC. (D) Receiver operating characteristic (ROC) curve analysis of tRF‐His‐GTG‐1 levels in pEVs from patients with acute COVID‐19 (with different disease severities and clinical outcomes). (E) tRF‐His‐GTG‐1 levels in pEVs positively correlated with LD accumulation in neutrophils from patients with COVID‐19. (F) tRF‐His‐GTG‐1 levels were elevated in neutrophils from patients with acute COVID‐19 (with different disease severities and clinical outcomes) and LC. (G) tRF‐His‐GTG‐1 levels in human neutrophils negatively correlated with RAB7A expression. (H,I) Electroporation‐mediated loading of tRF‐His‐GTG‐1 mimics into pEVs followed by coculture with human neutrophils resulted in increased intracellular tRF‐His‐GTG‐1 levels (left panel) and altered expression of lipophagy‐associated proteins as assessed by immunoblotting (right panel). (I) TEM showed that tRF‐His‐GTG‐1–overexpressing neutrophils accumulated abundant LDs clustered around lysosomes. (J) Immunofluorescence assay showed that tRF‐His‐GTG‐1 overexpression induced LD accumulation with reduced colocalization with lysosomes (LAMP1⁺), consistent with lipophagy impairment. Scale bars: 5 µm (immunofluorescence), 500 nm (TEM). All experiments were performed in triplicate. The data are presented as mean ± SD. Statistical significance was assessed using the Kruskal–Wallis test with Dunn's post hoc correction for multiple groups, and the Mann–Whitney U ‐test or unpaired two‐tailed Student's t ‐test for two‐group comparisons, as described in the Experimental Section. ** p < 0.01, *** p < 0.005. ns, nonsignificant. Densitometric analysis of immunoblotting is provided in Supporting Information . Abbreviations: HC, healthy controls; LC, patients with long COVID; Mod, patients with moderate COVID‐19; RC, patients who recovered from COVID‐19.

Article Snippet: Human primary neutrophils (2 × 106 cells per reaction) were transiently transfected with 500 ng DNA of the RAB7A expression plasmid (OriGene, RC201776) or an empty control vector using the Neon Transfection System Kit (Thermo Fisher Scientific, MPK1025) according to the manufacturer's instructions.

Techniques: Activation Assay, Quantitative RT-PCR, Derivative Assay, Recombinant, Infection, Expressing, Electroporation, Western Blot, Immunofluorescence, Over Expression, MANN-WHITNEY, Two Tailed Test

Journal: Advanced Science

Article Title: Extracellular Vesicle‐Delivered tRF‐His‐GTG‐1 Reprograms Neutrophil Lipophagy and Triggers Inflammation in COVID‐19

doi: 10.1002/advs.202508695

Figure Lengend Snippet: tRF‐His‐GTG‐1 inhibition restores lipophagy and suppresses NET formation and inflammation in COVID‐19 neutrophils. (A) Immunofluorescence analysis showed that treatment with a tRF‐His‐GTG‐1 inhibitor restored LD–lysosome (LAMP1⁺) colocalization, which was impaired by spike protein and pEV spike . (B) Immunoblotting revealed that the tRF‐His‐GTG‐1 inhibitor reversed spike protein– and pEV spike ‐induced RAB7A suppression and TLR8‐mTOR activation, leading to reduced expression of PLIN2, phosphorylated p47 PHOX , MPO, and citH3. (C–E) The tRF‐His‐GTG‐1 inhibitor suppressed LD accumulation, NET formation, and proinflammatory cytokine production (IL‐1β and IL‐8) induced by spike protein and pEV spike , as shown by (C) immunofluorescence and (D,E) quantification by flow cytometry and ELISA. (F–I) Neutrophils isolated from patients with COVID‐19 were treated ex vivo with the tRF‐His‐GTG‐1 inhibitor (200 µ m ), resulting in (F) enhanced colocalization of LDs and lysosomes (LAMP1⁺), (G) decreased NET DNA release, and (H) reduced LD accumulation. (I) Paired analysis of neutrophils from patients with COVID‐19 ( n = 27) treated ex vivo with a tRF‐His‐GTG‐1 inhibitor or a control tsRNA inhibitor showed significantly reduced LD levels, NET DNA release, and secretion of IL‐1β and IL‐8. Scale bars in immunofluorescence images represent 5 µm. All experiments were performed in triplicate, and data are presented as mean ± SD. Statistical significance was assessed using the Kruskal–Wallis test with Dunn's post hoc correction for multiple groups, and the Mann–Whitney U ‐test or unpaired two‐tailed Student's t ‐test for two‐group comparisons, as described in the Experimental Section. * p < 0.05, ** p < 0.01, *** p < 0.005. Densitometric analysis of immunoblotting is provided in Supporting Information .

Article Snippet: Human primary neutrophils (2 × 106 cells per reaction) were transiently transfected with 500 ng DNA of the RAB7A expression plasmid (OriGene, RC201776) or an empty control vector using the Neon Transfection System Kit (Thermo Fisher Scientific, MPK1025) according to the manufacturer's instructions.

Techniques: Inhibition, Immunofluorescence, Western Blot, Activation Assay, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Isolation, Ex Vivo, Control, MANN-WHITNEY, Two Tailed Test

Journal: Advanced Science

Article Title: Extracellular Vesicle‐Delivered tRF‐His‐GTG‐1 Reprograms Neutrophil Lipophagy and Triggers Inflammation in COVID‐19

doi: 10.1002/advs.202508695

Figure Lengend Snippet: Proposed model illustrating how pEVs and tRF‐His‐GTG‐1 regulate LD metabolism, NET formation, and proinflammatory cytokine production in neutrophils during acute COVID‐19 and long COVID. (A) In HC and RC, resting platelets release minimal pEVs containing tRF‐His‐GTG‐1. During acute and long COVID, SARS‐CoV‐2 spike protein induces platelet activation and promotes the release of tRF‐His‐GTG‐1‐enriched pEVs. These pEVs are taken up by neutrophils, wherein tRF‐His‐GTG‐1 activates endosomal TLR8 signaling and directly suppresses RAB7A mRNA expression. TLR8 activation upregulates mTOR and inhibits lipophagy, while RAB7A downregulation impairs LD–lysosome fusion. These effects collectively promote LD accumulation, ROS production, NET release, and increased secretion of IL‐1β and IL‐8. (B) Treatment with a tRF‐His‐GTG‐1 inhibitor restores RAB7A expression and suppresses TLR8–mTOR signaling, thereby reactivating lipophagy, facilitating LD clearance, and reducing NET formation and inflammatory cytokine production in neutrophils. Abbreviations: HC, healthy controls; LDs, lipid droplets; NETs, neutrophil extracellular traps; pEVs, platelet‐derived extracellular vesicles; RC, patients who had recovered from COVID‐19.

Article Snippet: Human primary neutrophils (2 × 106 cells per reaction) were transiently transfected with 500 ng DNA of the RAB7A expression plasmid (OriGene, RC201776) or an empty control vector using the Neon Transfection System Kit (Thermo Fisher Scientific, MPK1025) according to the manufacturer's instructions.

Techniques: Activation Assay, Expressing, Derivative Assay

Journal: Nature communications

Article Title: Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis.

doi: 10.1038/s41467-024-54324-9

Figure Lengend Snippet: Fig. 1 | Expression of Rab7a in melanoma versus non-melanoma cancer cells, correlation with TPC2 expression, and interaction of Rab7a with TPC2. a Gene expression profile (qPCR) of Rab7a in human melanoma lines compared to differ- ent non-melanoma cancer lines. Error bars represent mean values ± SEM. Data points represent biological replicates. b Protein levels corroborating mRNA expression data in melanoma lines (compared to the breast cancer line MDA-MB- 231). Shown are biological replicates, each. c Gene expression profile (qPCR) of Rab7b in human melanoma lines compared to different non-melanoma cancer lines. Data points show biological replicates. Error bars represent mean values ± SEM. Data points represent biological replicates. d Melanoma lines showing strong expression correlation between Rab7a and TPC2 but not TPC1. e Gene expression profile (qPCR) showing relative expression of TPCs in human melanoma lines

Article Snippet: The following antibodies were used for human Rab7 protein from Cell Signaling Technology: 2094S and 9367S, binding around residues Asp193 and Glu188, respectively, corresponding to Exon 6.

Techniques: Expressing, Gene Expression

Journal: Nature communications

Article Title: Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis.

doi: 10.1038/s41467-024-54324-9

Figure Lengend Snippet: Fig. 3 | Effect of Rab7 inhibitor on TPC2 activity and physical interaction of Rab7a with TPC2. a, b Inhibition of PI(3,5)P2 evoked currents in endolysosomes (EL), expressing hTPC2 alone or with Rab7a, using the Rab7-inhibitor CID1067700. Shown are representative current density-voltage relationships of enlarged EL, expressing hTPC2WT + hRab7WT or hTPC2WT alone, activated with 1 µM PI(3,5)P2 followed by application of CID1067700 (diff. conc.) and 1 mM ATP (max. effect). c Statistical summary of data as shown in (a, b) at −100 mV. Each dot represents a single current density value measured from one EL. Data were tested for statistical significance with one-way ANOVA test followed by Tukey’s post-test (***p < 0.001, ****p < 0.0001, n = 3). d Cartoon showing CRISPR/Cas9 strategy to knockout TPCN2 in the SK-MEL-5 cell line. e qPCR data showing relative expression of TPC2 in WT and TPC2 KO SK-MEL-5 (n = 3). f Statistical summary of data (average current densities at −100 mV) as shown in (g) (n = 6). g Representative current density-voltage relationships from −100 to +100 mV showing basal, TPC2-A1-P (20 µM) activated and ATP (1 mM) blocked currents.

Article Snippet: The following antibodies were used for human Rab7 protein from Cell Signaling Technology: 2094S and 9367S, binding around residues Asp193 and Glu188, respectively, corresponding to Exon 6.

Techniques: Activity Assay, Inhibition, Expressing, CRISPR, Knock-Out

Journal: Nature communications

Article Title: Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis.

doi: 10.1038/s41467-024-54324-9

Figure Lengend Snippet: Fig. 6 | Expression of MITF and GSK3β in different melanoma lines and effects of Rab7a or TPC2 KOor small molecule blockers. a Representative Western blots for MITF and Rab7 protein expression in different melanoma lines and in the breast cancer line MDA-MB-231, normalized to Vinculin. b, c Correlation plot for MITF/Rab7 expression (b) and statistical analysis of experiments as shown in (a) (mean values ± SEM, n = 5–8). Data points represent biological replicates (c). d Representative images of sections from healthy lymphnode (male, abdomen) and melanoma lymphnode metastasis (male, iliacal) samples stained with hMITF antibody (IHC). Scale bars = 5 mm. e IHC evaluation was carried out considering the percentage of stained tumor cells. Statistical significance was assessed by two- tailed unpaired t-test, *p = 0.0125 (mean ± SD). One dot corresponds to one independent human donor (n = 10 for each condition). Genetic knockout of either Rab7a (f, g) or TPC2 (h, i) in SK-MEL-5 cells shows reduction in the protein levels of

Article Snippet: The following antibodies were used for human Rab7 protein from Cell Signaling Technology: 2094S and 9367S, binding around residues Asp193 and Glu188, respectively, corresponding to Exon 6.

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