Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A) Lonp1 expression in the UMAP embedding of cells from embryonic lungs from E12.5 and E15.5 as assayed by scRNA-seq from Zepp. et al. 2021. (B) RNA levels of Lonp1 in Tbx4rtTA;tetOcre;Lonp1 and control at E13.5. n=4 for each group. (C) Lung morphology and H&E staining of Tbx4rtTA;tetOcre;Lonp1 and control at P39. Scale bars, 1 mm for whole left lobe. 100 μm for H&E. (D) RNA levels of Lonp1 in Shhcre;Lonp1 and control at E13.5. n=4 for each group. (E) In situ hybridization for Sox2 RNAs. Scale bars, 50 μm. (F) In situ hybridization for Sox9 RNAs. Scale bars, 50 μm. (G) Immunofluorescent labeling of SOX2 and SOX9 proteins in Shhcre;Lonp1 and control at E13.5. Scale bars, 100 μm. (H) Mitochondria ROS quantification by MitoSOX in Shhcre;Lonp1 and control at E13.5, followed by flow cytometry analysis. (I-J) Immunofluorescent labeling of FOXA1 in Shhcre;Lonp1 and control at E13.5. Quantifications were shown in (J). n=3 for each group. Scale bars, 50 μm. (K-L) Immunofluorescent labeling of FOXA2 in Shhcre;Lonp1 and control at E13.5. Quantifications were shown in (L). n=3 for each group. Scale bars, 50 μm. (M) qPCR of representative genes in SHH and FGF10 signaling at E13.5. n=4 for each group. (N) In situ hybridization for Fgf10 RNAs at E12.5. Scale bars, 50 μm. (O) Cleaved Caspase3 staining was not found neither in control nor in mutant naïve epithelium. Scale bars, 50 μm.

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Expressing, Control, Staining, In Situ Hybridization, Labeling, Flow Cytometry, Mutagenesis

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A) Lung morphology of Shhcre;Lonp1 and control at E13.5. Branching tips were denoted by arrowheads. Scale bars, 500 μm. (B) Tip numbers of left lobes at E13.5 (n=6 for each group). (C) Whole mount staining of E-Cadherin at E11.5. Tip number reduction was apparent in the left lobe as denoted by arrowheads. Scale bars, 200 μm. (D-E) Protein levels of representative ETC components in E13.5 lungs as assayed by western blot. Quantifications were shown in (E). n=3 for each group. (F) Quantifications of mitochondria DNA copy number. n=10 for each group. (G) Gene expression levels measured by bulk RNA-seq from E13.5 lungs. (H) qPCR of ER and mitochondria related stress genes at E13.5. n=4 for each group. (I-J) ATF4 immunostaining in E13.5 lungs. E-Cadherin was co-stained to label the epithelium. Quantifications were shown in (J). n=4 for each group. Scale bars, 50 μm. (K-L) CDKN1A immunostaining in E13.5 lungs. E-Cadherin was co-stained to label the epithelium. Nuclear staining of CDKN1A was marked by arrowheads. Quantifications were shown in (L). n=4 for each group. Scale bars, 50 μm. (M-N) Epithelial cell proliferation measured by EdU labeling at E12.5. Epithelium was marked by dashed lines. Quantifications were shown in (N). n=4 for each group. Scale bars, 50 μm.

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Control, Staining, Western Blot, Gene Expression, RNA Sequencing, Immunostaining, Labeling

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A-B) Immunofluorescent labeling of club cells (anti-SCGB1A1) and ciliated cells (anti-FOXJ1) in the trachea of control and Shhcre;Lonp1 at E18.5. Quantifications were shown in (B). n=3 for each group. Scale bars, 50 μm. (C-D) Immunofluorescent labeling of basal cells by TRP63 and KRT5 at E18.5. Quantifications of luminal staining of KRT5 were shown in (D). n=3 for each group. Scale bars, 50 μm. (E) Schematic of experimental procedure used to adult inactivation of Lonp1 . (F-G) Immunofluorescent labeling of club and ciliated cells in the trachea and lung of control and Sox2creER;Lonp1 . The magnified regions of the lung airway were marked by the yellow boxes in the middle image and shown in the right. Quantifications of the trachea staining were shown in (G). n=3 for each group. Scale bars, 50 μm for trachea in the left, 200 μm for lung in the middle and 20 μm for zoomed pictures in the right. (H-J) Immunofluorescent labeling of basal cells in the trachea and lung of control and Sox2creER;Lonp1 . The magnified regions of the ectopic basal-like cells in the mutant and the corresponding control in (I) were marked by the yellow boxes in (H). Quantifications of the luminal staining of KRT5 in trachea were shown in (J). n=3 for each group. Scale bars, 30 μm for trachea in the left, 500 μm for lung in the middle and 20 μm for zoomed pictures in the right

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Labeling, Control, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A) Lonp1 expression in the UMAP embedding of cells from adult trachea assayed by scRNA-seq from Montoro. et al. 2018. (B) Lonp1 RNA detection by RNAscope, counterstained with club, ciliated and basal cell markers. Scale bars, 20 μm. (C-D) Immunofluorescent labeling of basal cells in the trachea and lung of control and Sox2creER;Lonp1 at D30 post inactivation. The magnified regions of the ectopic basal-like cells marked by the yellow boxes in the mutant lung and the corresponding control were shown in the right. Quantifications of the luminal staining of KRT5 in trachea were shown in (D). n=3 for each group. Scale bars, 25 μm for trachea in the left, 100 μm for lung in the middle and 25 μm for zoomed pictures in the right.

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Expressing, RNA Detection, RNAscope, Labeling, Control, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A) Schematic of experimental procedure used to inactivate Lonp1 in club cells. (B) Most of the club cells were ablated in the lung airway 2 days post naphthalene injury. Scale bars, 50 μm. (C-D) Club cell proliferation at 5 days post injury as assayed by Mki67 staining. Quantifications were shown in (D). n=3 for each group. Scale bars, 50 μm. (E) Restoration of airway epithelium in both Scgb1a1creER;Lonp1 and control at 21 days post injury. Scale bars, 100 μm. (F) Quantification of club cells for each time point post naphthalene injury. (G) Schematic of experimental procedure used to inactivate Lonp1 in basal stem cells with a lineage marker. (H) Most of the club and ciliated cells were ablated in the trachea airway at 3 days post naphthalene injury. Scale bars, 20 μm. (I) Basal cell hyperplasia in Trp63creER;Lonp1;tdT and control trachea at 3 days post injury. Scale bars, 20 μm. (J) Regeneration of both club and ciliated cells in the trachea airway at 9 days post injury. Scale bars, 20 μm. (K) Basal cells were regressed to the basement of the trachea airway at 9 days post injury. Scale bars, 20 μm. (L) The regenerated airway epithelium was derived from basal cells as revealed by the global expression of lineage marker tdTomato. Scale bars, 20 μm. (M) Quantification of basal cells at D3 and D9 post naphthalene injury. (N) Quantification of club and ciliated cells at D3 and D9 post naphthalene injury.

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Staining, Control, Marker, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A) Schematic of experimental procedure used to analyze apoptosis in Sox2creER;Lonp1 . (B) Immunostaining for cleaved Caspase3 in the airway epithelium of control and Sox2creER;Lonp1 at 14 days post inactivation. Apoptotic cells were denoted by yellow arrows. The magnified regions of the airway were marked by the white stars and shown in the upper right. Scale bars, 50 μm. (C) Ratio of the apoptotic cells in the airway quantified for each time point. (D-E) Co-staining of cleaved Caspase3 with each cell marker for ciliated, basal and club cells at D14 and D22. Quantifications of co-detection at D14 were shown in (E). n=3 for each group. Scale bars, 20 μm. (F) Schematic of experimental procedure used to inactivate Lonp1 in ciliated cells. (G-H) Staining of cleaved Caspase3 in ciliated cells at D18. Quantifications of co-detection at multiple time points were shown in (H). n=3 for each group. Scale bars, 20 μm. (I) Schematic of experimental procedure used for continuous tamoxifen administration in Foxj1creER;Lonp1 . (J-L) Immunofluorescent labeling of club and ciliated cells in the lung of control and Foxj1creER;Lonp1 at D0, 30 and D60 post Lonp1 inactivation. Quantification of ciliated and club cell numbers at denoted time point were shown in (K) and (L), respectively. n=3 for each group. Scale bars, 20 μm.

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Immunostaining, Control, Staining, Marker, Labeling

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A) Schematic of experimental procedure used to isolate airway epithelial cells for scRNA-seq. (B) UMAP embedding of integrated cells from control and Sox2creER;Lonp1 . (C) Expression signatures of marker genes in each cell type. (D) UMAP embedding of integrated cells colored by control and Sox2creER;Lonp1 . (E) UMAP embedding of integrated cells with higher resolution. (F) Expression signatures of marker genes in each defined cell type. (G) Ratio of normalized cell numbers from control and Lonp1 mutant in each cell type. (H) Differentially enriched pathways between control and mutant airway cells analyzed by gene set enrichment analysis. Cell types were ordered horizontally by the relative proportions of control and mutant cell numbers, as denoted by the right triangles on the top. (I) Single cell enrichment scores of the ISR pathways projected onto UMAP. Cells exclusively identified in the mutant airway were marked by red dashed lines. (J) Gene expression features of representative factors in the ISR pathway. Cells exclusively identified in the mutant airway were marked by red dashed lines. (K) Distribution of enrichment scores for three branches of ER stress pathway in control and mutant cells. Wilcox test was used for statistical assay. (L) Schematic of experimental procedure used to analyze the ISR activation in Sox2creER;Lonp1 . (M) Co-immunostaining of ATF4 with FOXJ1 in the airway epithelium of control and Sox2creER;Lonp1 at 10 days post inactivation. Scale bars, 50 μm. (N) Ratio of the ATF4+ ciliated cells in the airway quantified for each time point. (O) Quantifications of ATF4 co-staining with ciliated, club and basal cell markers at 10 days post inactivation. n=3 for each group. (P) Schematic of experimental procedure used to assay airway phenotypes in Sox2creER;Lonp1;Atf4 . (Q-R) Immunofluorescent labeling of apoptotic signals on top of FOXJ1 in the trachea of control and relative mutants at 14 days post inactivation. Quantifications were shown in (R). n=3 for each group. Scale bars, 20 μm. (S-T) Immunofluorescent labeling of club and ciliated cells in the trachea of control and relative mutants at 30 days post inactivation. Quantifications were shown in (T). n=3 for each group. Scale bars, 20 μm. (U-V) Immunofluorescent labeling of basal cells in the trachea of control and relative mutants at 30 days post inactivation. Quantifications were shown in (V). n=3 for each group. Scale bars, 20 μm.

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Control, Expressing, Marker, Mutagenesis, Gene Expression, Activation Assay, Immunostaining, Staining, Labeling

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A) Schematic of experimental procedure used to analyze airway phenotypes in Sox2creER;Lonp1;Ddit3 . (B-C) Immunofluorescent labeling of club and ciliated cells in the trachea of control and relative mutants at 30 days post inactivation. Quantifications were shown in (C). n=3 for each group. Scale bars, 20 μm. (D-E) Immunofluorescent labeling of basal cells in the trachea of control and relative mutants at 30 days post inactivation. Quantifications were shown in (E). n=3 for each group. Scale bars, 20 μm. (F) RNA levels of Lonp1 in control, Lonp1 or Atf4 single mutants and Lonp1;Atf4 double mutants as assayed by qPCR. n=3 for each group. (G) RNA levels of Lonp1 in control, Lonp1 or Ddit3 single mutants and Lonp1;Ddit3 double mutants as assayed by qPCR. n=3 for each group.

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Labeling, Control

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A) Schematic of experimental procedure used to perform influenza infection to Sox2creER;Lonp1;tdT and the corresponding control. (B) Histological morphology of virus injured lungs measured by H&E staining, and the lineage tracing of KRT5+ airway stem cells in the adjacent sections on the right. Severe viral injury led to drastic loss of AT1 cells, which was evidenced by the absence of PDPN staining. Scale bars, 1 mm. (C) Magnified regions marked by yellow boxes in (B), which showed the stalled KRT5+ basal cells in the airway of Sox2creER;Lonp1;tdT . Scale bars, 50 μm. (D) Percentage of viral damaged areas covered by KRT5+ basal cells in controls and Lonp1 mutants. n>=5 for each group. (E) Number of KRT5+ basal cells in the injured airways of controls and Lonp1 mutants. n>=5 for each group.

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Infection, Control, Virus, Staining

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A) Body weight loss of control and Sox2creER;Lonp1 post influenza infection quantified for each time point. n=3 for each group. (B) Viral loads in control and Sox2creER;Lonp1 lungs at 6 days post infection, as quantified by qPCR. Two pairs of primers targeting viral genes M1 and NS1 , respectively, were used for analysis. n=4 for each group.

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Control, Infection

Journal: bioRxiv

Article Title: Context-dependent functions of mitochondria protein quality control in lung

doi: 10.1101/2022.12.08.519642

Figure Lengend Snippet: (A-B) Immunofluorescent labeling of ATF4 with ectopic KRT5+ basal cells in the airway of control and Sox2creER;Lonp1 at 14 days post influenza infection. Quantifications were shown in (B). n=3 for each group. Scale bars, 20 μm. (C) Cleaved Caspase3 staining was not found neither in control nor in mutant airways. Scale bars, 20 μm. (D-E) Immunofluorescent labeling of CDKN1A with ectopic KRT5+ basal cells in control and Sox2creER;Lonp1 at 14 days post influenza infection. Quantifications were shown in (E). n=3 for each group. Scale bars, 20 μm. (F) Schematic of experimental procedure used to perform influenza infection to Sox2creER;Lonp1;Atf4 and Sox2creER;Lonp1;Atf4/+ . (G-I) Immunofluorescent labeling of KRT5 at 14 days post influenza infection. Magnified regions marked by yellow boxes in (G) were shown in (H), percentage of KRT5+ areas in the damaged alveoli were quantified in (I). n=4 for each group. Scale bars, 1 mm in (G) and 100 μm in (H). (J) LONP1-mediated mitochondria protein quality control plays an array of essential roles in regulating airway epithelial cell proliferation, differentiation, survival and migration in a context dependent manner. Cartoons were created with BioRender.com .

Article Snippet: The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000).

Techniques: Labeling, Control, Infection, Staining, Mutagenesis, Migration

Journal: Redox Biology

Article Title: Endothelial Lon protease 1 facilitates the redox balance to prevent glomerulosclerosis by acting on superoxide dismutase 2 ubiquitination

doi: 10.1016/j.redox.2025.103929

Figure Lengend Snippet: LONP1 is downregulated in glomerulosclerotic kidney tissues and damaged endothelial cells. A) IF staining of LONP1 in the normal human and CKD patient kidneys. Scale bar, 10 μm. B) IF staining of LONP1 in the sham and 5/6Nx mice kidneys. Scale bar, 10 μm. C) RNA sequencing analysis showed the mRNA level of LONP1 in the kidney of sham and 5/6Nx mice ( n = 5). D) IF staining of LONP1 and CD31 in the normal human kidneys. LONP1 (red), CD31 (green), DAPI (blue), and co-localized position (white arrows). Scale bar, 10 μm. E, F) Humphreyslab database ( http://humphreyslab.com/SingleCell/ ) showed the expression of LONP1 in various cell populations of healthy human kidney tissues. G, I) LONP1 expression of the MAECs induced by Ang II at concentration of 0, 0.01, 0.1, 1 μM was determined by Western blot. GAPDH was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. H, J) LONP1 expression of the MAECs induced by ALD at concentration of 0, 100, 300, 500 nM was determined by Western blot. GAPDH was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. K, L) LONP1 expression of the MAECs induced by H 2 O 2 at concentration of 0, 100, 200, 300 μM was determined by Western blot. GAPDH was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. M, O, P) Quantification of the MFI of DCF (indicating total reactive oxygen species [ROS]; n = 3) and mitoSOX (indicating mitochondrial ROS [mtROS]; n = 3) using flow cytometry in glomerular vascular endothelial cells extracted from WT and hetero cKO mice (M), HAECs (O), and MAECs (P). N) MDA level in the urine of WT and hetero cKO mice after 5/6Nx ( n = 7–9). EC, Endothelial cell; CKD, chronic kidney disease; 5/6Nx, 5/6 nephrectomy; Ang II, Angiotensin; ALD, Aldosterone; H 2 O 2 , hydrogen peroxide; mtROS, mitochondrial ROS; mGEnCs, Mouse Glomerular Endothelial Cells; MAECs, Mouse aortic endothelial cells; HAECs, Human aortic endothelial cells.

Article Snippet: Cells were transfected with LONP1 or SOD2 plasmids or an shRNA targeting LONP1 using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Halethorpe, MD; 11668019) according to the manufacturer's instructions and then stimulated with 0.1 μM angiotensin II (Ang II) (Sigma-Aldrich, St. Louis, MO, USA; 4474-91-3) or control buffer for 24 h. The human LONP1 shRNA (sc-97290-SH) was purchased from Santa Cruz, and the mouse LONP1 shRNA (sequence: GCTGCATACAAGATCGTAA) was obtained from RiboBio (Guangzhou, China).

Techniques: Staining, RNA Sequencing, Expressing, Concentration Assay, Western Blot, Control, Flow Cytometry

Journal: Redox Biology

Article Title: Endothelial Lon protease 1 facilitates the redox balance to prevent glomerulosclerosis by acting on superoxide dismutase 2 ubiquitination

doi: 10.1016/j.redox.2025.103929

Figure Lengend Snippet: Endothelial LONP1 directly binds to SOD2 to alleviate oxidative stress. A, B) Western blot of LONP1, SOD1, SOD2, and SOD3 in primary HAECs transfected with NC plasmid, 300, 600, and 800 ng shRNA of LONP1 for 24 h. β-Actin was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. C, D) Western blot of LONP1, SOD1, SOD2, and SOD3 in MAECs transfected with NC plasmid, 300, 600, and 800 ng shRNA of LONP1 for 24 h. GAPDH was used as a control of LONP1, SOD1 and SOD2, β-Actin was used as a control of SOD3 ( n = 3). Dot plots represent quantitative densitometric data from Western blot. E, F) Western blot of LONP1 and SOD2 in glomerular vascular endothelial cells extracted from WT and hetero cKO mice. GAPDH was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. G, H) Western blot of LONP1 and SOD2 in primary HAECs transfected with NC plasmid, 200, and 400 ng LONP1 overexpression plasmid for 24 h. β-Actin was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. I, J) Representative Western blot of LONP1 and SOD2 in MAECs transfected with NC plasmid, 50, 100, and 200 ng LONP1 overexpression plasmid for 24 h. GAPDH was used as a control ( n = 4). Dot plots represent quantitative densitometric data from Western blot. K, L) Representative Western blot of SOD2 in MAECs transfected with NC plasmid and LONP1 overexpression plasmid, and treated with CHX for 0, 2, 4, 8, 12 h. β-Actin was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. M) Ubiquitination experiment of 293T cells transfected with LONP1 overexpression plasmid, SOD2 overexpression plasmid, and Ub plasmid. N) Ubiquitination experiment of 293T cells transfected with shRNA for LONP1, SOD2 overexpression plasmid, and Ub plasmid. O) Molecular interactions between the LONP1 and SOD2 by Molecular Dynamics Analysis. P, Q) Western blot of SOD2 in MAECs transfected with NC plasmid and SOD2 overexpression plasmid. GAPDH was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. R, S) Co-IP analysis of interaction between LONP1 and SOD2 in MAECs. T-V) Physical interaction between LONP1 and SOD2 protein performed by MST (T), SPR (U), and BLI (V). W) IF co-localization analysis of LONP1 and SOD2 in primary HAECs. LONP1 (red), SOD2 (green), DAPI (blue), and co-localized position (White dashed box). Scale bar, 10 μm. X) IF co-localization analysis of LONP1 and SOD2 in MAECs. LONP1 (green), SOD2 (red), DAPI (blue), and co-localized position (White dashed box). Scale bar, 10 μm. Y) Co-IP analysis of interaction between LONP1-Frag3 and SOD2 in MAECs. Z) Co-IP analysis of interaction between LONP1-MUT and SOD2 in MAECs. AA) Molecular interactions between the LONP1-MUT and SOD2 by Molecular Dynamics Analysis. NC, Negative Control; LONP1-Frag3, LONP1-Fragment3; LONP-MUT, LONP1-mutant; mGEnCs, Mouse Glomerular Endothelial Cells; MAECs, Mouse aortic endothelial cells; HAECs, Human aortic endothelial cells.

Article Snippet: Cells were transfected with LONP1 or SOD2 plasmids or an shRNA targeting LONP1 using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Halethorpe, MD; 11668019) according to the manufacturer's instructions and then stimulated with 0.1 μM angiotensin II (Ang II) (Sigma-Aldrich, St. Louis, MO, USA; 4474-91-3) or control buffer for 24 h. The human LONP1 shRNA (sc-97290-SH) was purchased from Santa Cruz, and the mouse LONP1 shRNA (sequence: GCTGCATACAAGATCGTAA) was obtained from RiboBio (Guangzhou, China).

Techniques: Western Blot, Transfection, Plasmid Preparation, shRNA, Control, Over Expression, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Negative Control, Mutagenesis

Journal: Redox Biology

Article Title: Endothelial Lon protease 1 facilitates the redox balance to prevent glomerulosclerosis by acting on superoxide dismutase 2 ubiquitination

doi: 10.1016/j.redox.2025.103929

Figure Lengend Snippet: Heterozygous knockout of endothelial cell LONP1 aggravates glomerulosclerosis and inflammation in CKD induced by 5/6Nx. A, B) Analysis of BUN (A) and Cr (B) ( n = 13–17). C) ELISA of urinary microalbumin ( n = 7–9). D) Coomassie blue staining of representative urine samples by sodium dodecyl sulfate polyacrylamide gel electrophoresis. E) Masson's trichrome staining of WT and hetero cKO mice after 5/6Nx ( n = 7–9, Scale bar, 20 μm). F, H) Fibrotic area of glomerulus (F) and tubules (H) statistics of Masson's trichrome staining in WT and hetero cKO mice after 5/6Nx ( n = 7–9). G) PAS staining of WT and hetero cKO mice after 5/6Nx ( n = 7–9, Scale bar, 20 μm). Pathological changes of glomerulosclerosis (red arrows). I) Systolic blood pressure of WT and hetero cKO mice after 5/6Nx 4 w, 8 w, and 12 w detected by tail-cuff ( n = 8–15). J) IF staining of Fibronectin in WT and hetero cKO mice after 5/6Nx ( n = 5, Scale bar, 20 μm). K) Relative fluorescence intensity statistics of IF staining ( n = 5). L) IHC staining of F4/80 in WT and hetero cKO mice after 5/6Nx ( n = 5, Scale bar, 50 μm). M) IHC semi-quantitative IOD analysis of F4/80 ( n = 5). N, O) The IL-6 levels of serum (N) ( n = 7–8) and urine (O) ( n = 7–9) in WT and hetero cKO mice after 5/6Nx were detected by ELISA. IOD, Integral Optical Density.

Article Snippet: Cells were transfected with LONP1 or SOD2 plasmids or an shRNA targeting LONP1 using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Halethorpe, MD; 11668019) according to the manufacturer's instructions and then stimulated with 0.1 μM angiotensin II (Ang II) (Sigma-Aldrich, St. Louis, MO, USA; 4474-91-3) or control buffer for 24 h. The human LONP1 shRNA (sc-97290-SH) was purchased from Santa Cruz, and the mouse LONP1 shRNA (sequence: GCTGCATACAAGATCGTAA) was obtained from RiboBio (Guangzhou, China).

Techniques: Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Polyacrylamide Gel Electrophoresis, Fluorescence, Immunohistochemistry

Journal: Redox Biology

Article Title: Endothelial Lon protease 1 facilitates the redox balance to prevent glomerulosclerosis by acting on superoxide dismutase 2 ubiquitination

doi: 10.1016/j.redox.2025.103929

Figure Lengend Snippet: Knockdown of LONP1 promotes Ang II-induced mitochondrial dysfunction and inflammatory damage in vascular endothelial cells. A) OCR of primary HAECs transfected with LONP1 overexpression plasmid and treated with Ang II ( n = 6). B) Quantification of basal respiratory, maximal respiratory, and ATP production in Fig. A. C) OCR of MAECs transfected with LONP1 overexpression plasmid and treated with Ang II ( n = 6). D) Quantification of basal respiratory, maximal respiratory, and ATP production in Fig. C. E, F) IF staining of MitoTracker in primary HAECs (E) and MAECs (F) transfected with LONP1 overexpression plasmid and treated with Ang II ( n = 3, Scale bar, 10 μm). G, H) Relative fluorescence intensity of MitoTracker in primary HAECs (G) and MAECs (H) transfected with LONP1 overexpression plasmid and treated with Ang II ( n = 3). I) mitochondrial DNA (mtDNA) was assessed by quantitative polymerase chain reaction, and the number of mitochondrial genome copies (detected by the ND1 primer) were normalized to 18S ribosomal RNA ( n = 6). J) mRNA expression of genes encoded by the mitochondrial genome in MAECs transfected with LONP1 overexpression plasmid and treated with Ang II ( n = 3). K) OCR of primary HAECs transfected with shRNA of LONP1 and treated with Ang II ( n = 6). L) Quantification of basal respiratory, maximal respiratory, and ATP production in Fig. K. M) OCR of MAECs transfected with shRNA of LONP1 and treated with Ang II ( n = 6). N) Quantification of basal respiratory, maximal respiratory, and ATP production in Fig. M. O) Relative fluorescence intensity of MitoTracker in primary HAECs transfected with shRNA of LONP1 and treated with Ang II ( n = 3). P, Q) IF staining of MitoTracker in primary HAECs (P) and MAECs (Q) transfected with shRNA of LONP1 and treated with Ang II ( n = 3, Scale bar, 10 μm). R) Relative fluorescence intensity of MitoTracker in MAECs transfected with shRNA of LONP1 and treated with Ang II ( n = 3). S, T) Western blot of CD31, VCAM-1, and ICAM-1 in primary HAECs transfected with shRNA of LONP1 for 24 h. β-Actin was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. U, V) Western blot of CD31, VCAM-1, and ICAM-1 in MAECs transfected with shRNA of LONP1 for 24 h. β-Actin was used as a control of ICAM-1, GAPDH was used as a control of CD31 and VCAM-1 ( n = 3). Dot plots represent quantitative densitometric data from Western blot. W) Heatmap of upregulated inflammatory factors between the Vehicle and shLONP1 groups. X) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Gene Ontology (GO) analysis showed that differential signaling pathways were identified by the differential genes between the Vehicle and shLONP1 groups. Y, Z) Western blot of CD31, VCAM-1, and ICAM-1 in immortalized HAECs transfected with LONP1 plasmid and treated with Ang II. β-Actin was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. AA, AB) Western blot of CD31, VCAM-1, and ICAM-1 in MAECs transfected with LONP1 plasmid and treated with Ang II. β-Actin was used as a control of CD31 and VCAM-1, GAPDH was used as a control of ICAM-1 ( n = 3). Dot plots represent quantitative densitometric data from Western blot. AC, AD) The IL-6 (AC) and TNF-α (AD) levels of cell medium in MAECs transfected with LONP1 plasmid and treated with Ang II were detected by ELISA ( n = 4).

Article Snippet: Cells were transfected with LONP1 or SOD2 plasmids or an shRNA targeting LONP1 using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Halethorpe, MD; 11668019) according to the manufacturer's instructions and then stimulated with 0.1 μM angiotensin II (Ang II) (Sigma-Aldrich, St. Louis, MO, USA; 4474-91-3) or control buffer for 24 h. The human LONP1 shRNA (sc-97290-SH) was purchased from Santa Cruz, and the mouse LONP1 shRNA (sequence: GCTGCATACAAGATCGTAA) was obtained from RiboBio (Guangzhou, China).

Techniques: Knockdown, Transfection, Over Expression, Plasmid Preparation, Staining, Fluorescence, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot, Control, Protein-Protein interactions, Enzyme-linked Immunosorbent Assay

Journal: Redox Biology

Article Title: Endothelial Lon protease 1 facilitates the redox balance to prevent glomerulosclerosis by acting on superoxide dismutase 2 ubiquitination

doi: 10.1016/j.redox.2025.103929

Figure Lengend Snippet: SOD2 supplementation alleviates glomerulosclerosis and inhibits the vascular endothelial cells inflammation response. A) Masson's trichrome staining in different groups ( n = 8–9, Scale bar, 50 μm). B, C) Fibrotic area of glomerulus (B) and tubules (C) statistics of Masson's trichrome staining in different groups ( n = 8–9). D) PAS staining in different groups ( n = 8–9, Scale bar, 50 μm). E) Systolic blood pressure in different groups detected by tail-cuff ( n = 8–10). F) ELISA of urinary microalbumin in different groups ( n = 8–10). G) Analysis of Cr in different groups ( n = 8–9). H) Relative fluorescence intensity statistics of IF staining of Fibronectin ( n = 5). I) IF staining of Fibronectin in different groups ( n = 5, Scale bar, 20 μm). J, K) Western blot of Fibronectin in different groups. GAPDH was used as a control ( n = 4). Dot plots represent quantitative densitometric data from Western blot. L) IHC staining of F4/80 in different groups ( n = 5, Scale bar, 50 μm). M) IHC semi-quantitative IOD analysis of F4/80 ( n = 5). N, O) Western blot of CD31, VCAM-1, and ICAM-1 in immortalized HAECs transfected with shRNA of LONP1 and treated with MnTBAP. β-Actin was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. P, Q) Western blot of CD31, VCAM-1, and ICAM-1 in MAECs transfected with shRNA of LONP1 and treated with MnTBAP. GAPDH was used as a control of CD31, β-Actin was used as a control of VCAM-1 and ICAM-1 ( n = 3). Dot plots represent quantitative densitometric data from Western blot. R, S) The IL-6 (R) and TNF-α (S) levels of cell medium in MAECs transfected with shRNA of LONP1 and treated with MnTBAP were detected by ELISA ( n = 3). T, U) Western blot of CD31, VCAM-1, and ICAM-1 in immortalized HAECs co-transfected with shRNA of LONP1 and SOD2 overexpression plasmid. β-Actin was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. V, W) Western blot of CD31, VCAM-1, and ICAM-1 in MAECs co-transfected with shRNA of LONP1 and SOD2 overexpression plasmid. GAPDH was used as a control of CD31, β-Actin was used as a control of VCAM-1 and ICAM-1 ( n = 3). Dot plots represent quantitative densitometric data from Western blot. X, Y) The IL-6 (X) and TNF-α (Y) levels of cell medium in MAECs co-transfected with shRNA of LONP1 and SOD2 overexpression plasmid were detected by ELISA ( n = 3). IOD, Integral Optical Density.

Article Snippet: Cells were transfected with LONP1 or SOD2 plasmids or an shRNA targeting LONP1 using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Halethorpe, MD; 11668019) according to the manufacturer's instructions and then stimulated with 0.1 μM angiotensin II (Ang II) (Sigma-Aldrich, St. Louis, MO, USA; 4474-91-3) or control buffer for 24 h. The human LONP1 shRNA (sc-97290-SH) was purchased from Santa Cruz, and the mouse LONP1 shRNA (sequence: GCTGCATACAAGATCGTAA) was obtained from RiboBio (Guangzhou, China).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Fluorescence, Western Blot, Control, Immunohistochemistry, Transfection, shRNA, Over Expression, Plasmid Preparation

Journal: Redox Biology

Article Title: Endothelial Lon protease 1 facilitates the redox balance to prevent glomerulosclerosis by acting on superoxide dismutase 2 ubiquitination

doi: 10.1016/j.redox.2025.103929

Figure Lengend Snippet: Knockdown of endothelial cell LONP1 facilitates Ang II-induced activation and proliferation of mesangial cells. A, B) Western blot of CyclinD1, CyclinA2 ( n = 6), and Fibronectin ( n = 3) in MCs induced by culture supernatant of MAECs transfected with LONP1 plasmid and treated with Ang II. β-Actin was used as a control. Dot plots represent quantitative densitometric data from Western blot. C, D) Representative micrographs with IF staining and the MFI of EdU in MCs induced by culture supernatant of MAECs transfected with LONP1 plasmid and treated with Ang II ( n = 3, Scale bar, 40 μm). E, F) Western blot of CyclinD1 and CyclinA2 in MCs induced by culture supernatant of MAECs transfected with shRNA of LONP1. GAPDH was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. G, H) Representative micrographs with IF staining and the mean fluorescence intensity of EdU in MCs induced by culture supernatant of MAECs transfected with shRNA of LONP1 and treated with Ang II ( n = 3, Scale bar, 40 μm). I, J) Western blot of CyclinD1 in MCs induced by culture supernatant of MAECs transfected with shRNA of LONP1 and treated with Ang II. β-Actin was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. K, L) Western blot of CyclinD1 in MCs induced by culture supernatant of MAECs co transfected with shRNA of LONP1 and SOD2 overexpression plasmid. GAPDH was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot.

Article Snippet: Cells were transfected with LONP1 or SOD2 plasmids or an shRNA targeting LONP1 using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Halethorpe, MD; 11668019) according to the manufacturer's instructions and then stimulated with 0.1 μM angiotensin II (Ang II) (Sigma-Aldrich, St. Louis, MO, USA; 4474-91-3) or control buffer for 24 h. The human LONP1 shRNA (sc-97290-SH) was purchased from Santa Cruz, and the mouse LONP1 shRNA (sequence: GCTGCATACAAGATCGTAA) was obtained from RiboBio (Guangzhou, China).

Techniques: Knockdown, Activation Assay, Western Blot, Transfection, Plasmid Preparation, Control, Staining, shRNA, Fluorescence, Over Expression

Journal: Redox Biology

Article Title: Endothelial Lon protease 1 facilitates the redox balance to prevent glomerulosclerosis by acting on superoxide dismutase 2 ubiquitination

doi: 10.1016/j.redox.2025.103929

Figure Lengend Snippet: Knockdown of endothelial LONP1 facilitates Ang II-induced podocytes injury. A) Representative images of TEM on MPCs induced by culture supernatant of MAECs ( n = 3, Scale bar, 2 μm and 1 μm). Mitochondria (red arrows), cytoplasmic vacuole (yellow arrows), and autophagosome (blue arrows). B-D) Western blot of Podocin and BCL2 in MPCs induced by culture supernatant of MAECs transfected with LONP1 plasmid and treated with Ang II. GAPDH was used as a control ( n = 3). Dot plots represent quantitative densitometric data from Western blot. E, F) Representative images for fluorescence-activated cell sorting (FACS) analysis after annexin V and PI staining in MPCs induced by culture supernatant of MAECs transfected with LONP1 plasmid and treated with Ang II. Quantitative analysis of apoptotic cells ( n = 3). G, H) Representative images for FACS analysis after annexin V and PI staining in MPCs induced by culture supernatant of MAECs transfected with shRNA of LONP1 and treated with Ang II. Quantitative analysis of apoptotic cells ( n = 3). I) Volcano plot of differentially expressed genes between vehicle and shLONP1 groups ( n = 3). J) KEGG pathway analysis of differentially expressed genes between two groups of MPCs induced by culture supernatant of MAECs transfected with shRNA of LONP1 and NC plasmid ( n = 3). K, L) Expression of differentially expressed genes between vehicle and shLONP1 groups of MPCs in apoptosis (K) and p53 signaling pathway (L) ( n = 3). M) Analysis of the percentage of foot process fusion in Fig. N ( n = 4). N) Representative images of TEM on renal tissues of WT and hetero cKO mice after 5/6Nx ( n = 4, Scale bar, 2 μm and 500 nm). O) Analysis of foot processes width in Fig. N ( n = 4). P) Analysis of the number of foot processes per 1 cm GBM in Fig. N ( n = 4).

Article Snippet: Cells were transfected with LONP1 or SOD2 plasmids or an shRNA targeting LONP1 using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Halethorpe, MD; 11668019) according to the manufacturer's instructions and then stimulated with 0.1 μM angiotensin II (Ang II) (Sigma-Aldrich, St. Louis, MO, USA; 4474-91-3) or control buffer for 24 h. The human LONP1 shRNA (sc-97290-SH) was purchased from Santa Cruz, and the mouse LONP1 shRNA (sequence: GCTGCATACAAGATCGTAA) was obtained from RiboBio (Guangzhou, China).

Techniques: Knockdown, Western Blot, Transfection, Plasmid Preparation, Control, Fluorescence, FACS, Staining, shRNA, Expressing

Journal: PLOS Computational Biology

Article Title: An integrative analysis reveals the mechanism of plastic stabilizers inducing breast cancer

doi: 10.1371/journal.pcbi.1014025

Figure Lengend Snippet: Visualization of the binding modes between three PSs (2,6-DTB, TBHQ, UV-328) and five core targets (GSK3B, MAPK14, PARP1, PIM1, TRDMT1). The 3D structures show the binding pockets, while 2D diagrams illustrate specific interaction types. The binding energies for the receptor-ligand complexes are provided in .

Article Snippet: Primary antibodies against MAPK14 (Cat# 14064–1-AP), PIM1 (Cat# 27196–1-AP), and TRDMT1 (Cat# 19221–1-AP) were purchased from Proteintech (USA).

Techniques: Binding Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: ATP-Dependent Lon Protease Contributes to Helicobacter pylori -Induced Gastric Carcinogenesis

doi: 10.1016/j.neo.2016.03.001

Figure Lengend Snippet: Lonp1 expression is induced by H. pylori infection in human gastric epithelial cells. (A) Heatmap showing the expression pattern of mitochondrial proteases in Kx1 and Kx2 H. pylori infection models. (B) Lonp1 mRNA levels in MKN28 gastric cells were indicated by qPCR in response to H. pylori infection (MOI = 50, NCTC 11637) at different time points. (C) Lonp1 mRNA levels in MKN28 gastric cells were induced by different MOIs of H. pylori NCTC 11637 for 4 hours. (D) Lonp1 mRNA levels in MKN28 gastric cells were induced by different H. pylori strains (MOI = 50, 26695, J99, BCM-300, and BCS 100) for 12 hours. (E) Lonp1 proteins that were indicated by western blot (left) and quantified (right) in MKN28 gastric cells were elevated by H. pylori infection (MOI = 50, NCTC 11637) at different time points. Data represent the mean ± SEM from three separate experiments. * P < .05.

Article Snippet: Lentiviral overexpression vector pLJM1-EGFP (19319) and pcDNA3 vector containing full-length HIF-1α (18949) were obtained from Addgene. pCMV6-XL4 vector containing full-length Lonp1 (SC117111) was purchased from Origene.

Techniques: Expressing, Infection, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: ATP-Dependent Lon Protease Contributes to Helicobacter pylori -Induced Gastric Carcinogenesis

doi: 10.1016/j.neo.2016.03.001

Figure Lengend Snippet: Induction of Lonp1 maintains mitochondrial function in response to low multiplicity of H. pylori infection. (A) Western blot indicating Lonp1 protein levels in MKN28 cells in response to low multiplicity of H. pylori infection (MOI = 50, NCTC 11637) and lentivrius-induced RNAi. (B-C) Mitochondrial mass, which was indicated by immunostaining of ATP5A (B), and mtDNA level (C) in MKN28 cells under low multiplicity of H. pylori infection for 24 hours (MOI = 50, NCTC 11637). (D-E) Basal and maximal mitochondrial oxygen rates (D) and cellular ATP levels (E) in control (sh-GFP) and Lonp1-deficient (sh-Lonp1) MKN28 cells in response to 24 hours of low multiplicity of H. pylori infection were measured. (F-G) Basal and maximal mitochondrial oxygen rates (F) and cellular ATP levels (G) in control (Con) and Lonp1-overexpressing (Lonp1) MKN28 cells. Data represent the mean ± SEM from three separate experiments. * P < .05.

Article Snippet: Lentiviral overexpression vector pLJM1-EGFP (19319) and pcDNA3 vector containing full-length HIF-1α (18949) were obtained from Addgene. pCMV6-XL4 vector containing full-length Lonp1 (SC117111) was purchased from Origene.

Techniques: Infection, Western Blot, Immunostaining, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: ATP-Dependent Lon Protease Contributes to Helicobacter pylori -Induced Gastric Carcinogenesis

doi: 10.1016/j.neo.2016.03.001

Figure Lengend Snippet: Low multiplicity of H. pylori infection results in glycolytic switch via Lonp1 induction. (A-B) Glucose incorporation (A) and lactate production (B) in control (sh-GFP) and Lonp1-deficient (sh-Lonp1) MKN28 cells in response to low multiplicity of H. pylori infection (MOI = 50, NCTC 11637) for 24 hours were indicated. (C-D) Glucose incorporation (C) and lactate production (D) in control (Con) and Lonp1-overexpressing (Lonp1) MKN28 cells were measured. Data represent the mean ± SEM from three separate experiments. * P < .05.

Article Snippet: Lentiviral overexpression vector pLJM1-EGFP (19319) and pcDNA3 vector containing full-length HIF-1α (18949) were obtained from Addgene. pCMV6-XL4 vector containing full-length Lonp1 (SC117111) was purchased from Origene.

Techniques: Infection, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: ATP-Dependent Lon Protease Contributes to Helicobacter pylori -Induced Gastric Carcinogenesis

doi: 10.1016/j.neo.2016.03.001

Figure Lengend Snippet: Lonp1 is required for low multiplicity of H. pylori -induced gastric cell proliferation. (A-B) Cell growth curves of control (sh-GFP) and Lonp1-deficient (sh-Lonp1) MKN28 cells in response to low multiplicity of H. pylori infection (MOI = 50, NCTC 11637) (A) and Lonp1-overexpressing MKN28 cells (B). (C-D) Images (C) and proliferation rates indicated by MTT assays (D) of control (sh-GFP) and Lonp1-deficient (sh-Lonp1) MKN28 cells in response to low multiplicity of H. pylori infection at day 5 (MOI = 50, NCTC 11637). (E-F) Images (E) and proliferation rates indicated by MTT assays (F) of control (Con) and Lonp1-overexpressing (Lonp1) MKN28 cells. Each group was compared with sh-GFP (A) or Con (B) at each time point in growth curve experiment. Data represent the mean ± SEM from three separate experiments. * P < .05.

Article Snippet: Lentiviral overexpression vector pLJM1-EGFP (19319) and pcDNA3 vector containing full-length HIF-1α (18949) were obtained from Addgene. pCMV6-XL4 vector containing full-length Lonp1 (SC117111) was purchased from Origene.

Techniques: Control, Infection

Journal: Neoplasia (New York, N.Y.)

Article Title: ATP-Dependent Lon Protease Contributes to Helicobacter pylori -Induced Gastric Carcinogenesis

doi: 10.1016/j.neo.2016.03.001

Figure Lengend Snippet: HIF-1α contributes to low MOI of H. pylori -induced Lonp1 induction and gastric cell proliferation. (A) Heatmap showing differential expression of known HIF-1α targets in Kx1 and Kx2 H. pylori infection models. (B-C) HIF-1α protein levels indicated by western blot (B) and HIF1a mRNA levels indicated by qPCR (C) in MKN28 gastric cells in response to low multiplicity of H. pylori infection at different time points (MOI = 50, NCTC 11637). (D) Hk2 and Pfkfb3 , two HIF-1α targets, in MKN28 cells were transcriptionally elevated by low multiplicity of H. pylori infection (MOI = 50, NCTC 11637) for 12 hours. (E-F) Lonp1 protein levels (E) and mRNA levels (F) in HIF-1α-overexpressing MKN 28 cells (left) or HIF-1α-knockdown MKN28 cells in response to low multiplicity of H. pylori infection for 12 hours (MOI = 50, NCTC 11637) (right). (G) Cell growth of control and HIF-1α-overexpressing MKN28 gastric cells (left) or HIF-1α-knockdown cells in response to low multiplicity of H. pylori infection (right). Data represent the mean ± SEM from three separate experiments. * P < .05.

Article Snippet: Lentiviral overexpression vector pLJM1-EGFP (19319) and pcDNA3 vector containing full-length HIF-1α (18949) were obtained from Addgene. pCMV6-XL4 vector containing full-length Lonp1 (SC117111) was purchased from Origene.

Techniques: Quantitative Proteomics, Infection, Western Blot, Knockdown, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: ATP-Dependent Lon Protease Contributes to Helicobacter pylori -Induced Gastric Carcinogenesis

doi: 10.1016/j.neo.2016.03.001

Figure Lengend Snippet: Schematic of molecular mechanism of Lonp1 induction and gastric carcinogenesis in response to H. pylori infection. Low multiplicity of H. pylori infection increases Lonp1 expression in gastric cells via HIF-1α regulation and other unknown mechanisms. Subsequently, excess Lonp1 results in mitochondrial restoration and metabolic switched toward glycolysis and contribute to gastric cell overproliferation and gastric carcinogenesis.

Article Snippet: Lentiviral overexpression vector pLJM1-EGFP (19319) and pcDNA3 vector containing full-length HIF-1α (18949) were obtained from Addgene. pCMV6-XL4 vector containing full-length Lonp1 (SC117111) was purchased from Origene.

Techniques: Infection, Expressing