Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Effect of AQP8 knockdown on hydrogen peroxide diffusion through the plasma membrane (NES), the outer mitochondrial membrane (IMS), and the inner mitochondrial membrane (MLS) of HeLa cells. ( A , D , G ) The left and right panels display representative frames extracted from videos illustrating the time course of H 2 O 2 diffusion into mock-transfected (Ctr) and AQP8-knockdown (siRNA AQP8) HeLa cells before and after the addition of 50 μM H 2 O 2 , respectively. The increase in HyPer7 fluorescence is shown in pseudocolor in the upper panel, with the scale indicated in the insert. ( B , E , H ) The time course of H 2 O 2 fluorescence in mock- and siRNA-transfected HeLa cells is presented, starting from the addition of 50 μM H 2 O 2 . Data represent the mean of at least three different experiments, with standard deviations omitted for clarity. ( C , F , I ) Computerized least squares regression analysis was employed to determine the k relative initial rate values (GraphPad Prism 4.00, 2003). The experimental points of the H 2 O 2 time course curves were fitted using a one-phase exponential association equation. a, p < 0.05 compared to Ctr (Student’s t -test).

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Knockdown, Diffusion-based Assay, Clinical Proteomics, Membrane, Transfection, Fluorescence

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Effect of double knockdown of AQP3/AQP6/AQP8 and S1R in the hydrogen peroxide diffusion through the plasma membrane (NES) of HeLa cells. ( A ) The left and right panels display representative frames extracted from videos illustrating the time course of H 2 O 2 diffusion into HeLa cells that were single knockdown for AQP (siRNA AQP3/AQP6/AQP8) and HeLa cells that were double knockdown for AQP and S1R (siRNA AQP3/AQP6/AQP8 +S1R) before and after the addition of 50 μM H 2 O 2 , respectively. The increase in HyPer7 fluorescence is shown in pseudocolor in the upper panel, with the scale indicated in the insert. ( B , D , F ) The time course of H 2 O 2 fluorescence in HeLa cells with a knockdown of AQP and in cells with a double knockdown of AQP and S1R is presented, starting from the addition of 50 μM H 2 O 2 . Data represent the mean of at least three different experiments, with standard deviations omitted for clarity. ( C , E , G ) Computerized least squares regression analysis was performed to determine the k relative initial rate values (GraphPad Prism 4.00, 2003). The experimental points from the time courses of H 2 O 2 curves were fitted using a one-phase exponential association equation. a, p < 0.05 compared to control (Student’s t -test).

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Knockdown, Diffusion-based Assay, Clinical Proteomics, Membrane, Fluorescence, Control

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Effect of double knockdown of AQP6/AQP8 and S1R on hydrogen peroxide diffusion through the inner mitochondrial membranes (MLS) of HeLa cells. ( A ) The left and right panels show representative frames extracted from videos displaying the time course of H 2 O 2 diffusion into HeLa cells that were single knockdown for AQP (siRNA AQP6/AQP8) and HeLa cells that were double knockdown for AQP and S1R (siRNA AQP6/AQP8 + S1R) before and after the addition of 50 μM H 2 O 2 , respectively. The increase in HyPer7 fluorescence is depicted in pseudocolor in the upper panel, with the scale shown in the insert. ( B , D ) The time course of H 2 O 2 fluorescence in HeLa cells knocked down for AQP and in those knocked down for both AQP and S1R is shown, starting from the addition of 50 μM H 2 O 2 . Data represent the mean of at least three different experiments, with standard deviations omitted for clarity. ( C , E ) Computerized least squares regression analysis was used to determine the k relative initial rate values (GraphPad Prism 4.00, 2003). The experimental points of the time courses of H 2 O 2 curves were fitted with a one-phase exponential association equation. a, p < 0.05 compared to control (Student’s t -test).

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Knockdown, Diffusion-based Assay, Fluorescence, Control

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Representative immunofluorescence confocal microscopy images of colocalization of AQP8 and AQP11 with TOMM20 in HeLa cells. ( A , C ) Green labeling indicates the presence of TOMM20, and red labeling indicates the expression of AQP8 or AQP11 (Hoechst (blue) counterstained nuclei). Yellow labeling shows colocalization signal of AQP8 or AQP11 with TOMM20. Scale bar, 5 μm. ( B , D ) The graph, measured in the white line position in panels ( A , C ), shows the fluorescence signals of AQP8 or AQP11 and TOMM20 staining.

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Immunofluorescence, Confocal Microscopy, Labeling, Expressing, Fluorescence, Staining

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: A possible schematic model of the functional AQPs in the plasma membrane and in the outer and inner mitochondrial membranes. AQP3, AQP6, and AQP8 are functionally active in the plasma membrane, AQP6 is functionally active in the outer mitochondrial membrane, while AQP6 and AQP8 are functionally active in the inner mitochondrial membrane. Arrows indicate the modulation of AQP8 by S1R, while the question mark indicates the uncertain modulation of AQP6.

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Functional Assay, Clinical Proteomics, Membrane

Journal: International Journal of Molecular Sciences

Article Title: The Role of Sensory Nerves in Dental Pulp Homeostasis: Histological Changes and Cellular Consequences after Sensory Denervation

doi: 10.3390/ijms25021126

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Laminin , Rabbit , Boster , PA1581.

Techniques:

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Effect of AQP11 knockdown on hydrogen peroxide diffusion through the plasma membrane (NES), the outer mitochondrial membrane (IMS), and the inner mitochondrial membrane (MLS) of HeLa cells. ( A , D , G ) The left and right panels display representative frames extracted from videos illustrating the time course of H 2 O 2 diffusion into mock-transfected (Ctr) and AQP11-knockdown (siRNA AQP11) HeLa cells before and after the addition of 50 μM H 2 O 2 , respectively. The increase in HyPer7 fluorescence is shown in pseudocolor in the upper panel, with the scale indicated in the insert. ( B , E , H ) The time course of H 2 O 2 fluorescence in mock- and siRNA-transfected HeLa cells is presented, starting from the addition of 50 μM H 2 O 2 . Data represent the mean of at least three different experiments, with standard deviations omitted for clarity. ( C , F , I ) Computerized least squares regression analysis was employed to determine the k relative initial rate values (GraphPad Prism 4.00, 2003). The experimental points of the H 2 O 2 time course curves were fitted using a one-phase exponential association equation.

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Knockdown, Diffusion-based Assay, Clinical Proteomics, Membrane, Transfection, Fluorescence

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Effect of double knockdown of AQP6/AQP11 and S1R on hydrogen peroxide diffusion through the outer mitochondrial membranes (IMS) of HeLa cells. ( A ) The left and right panels show representative frames extracted from videos displaying the time course of H 2 O 2 diffusion into HeLa cells that were single knockdown for AQP (siRNA AQP6/AQP11) and HeLa cells that were double knockdown for AQP and S1R (siRNA AQP6/AQP11 + S1R) before and after the addition of 50 μM H 2 O 2 , respectively. The increase in HyPer7 fluorescence is depicted in pseudocolor in the upper panel, with the scale shown in the inset. ( B , D ) The time course of H 2 O 2 fluorescence in HeLa cells knocked down for AQP and in those knocked down for both AQP and S1R is shown, starting from the addition of 50 μM H 2 O 2 . Data are the mean of at least three different experiments, with standard deviations omitted for clarity. ( C , E ) Computerized least squares regression analysis was used to determine the k relative initial rate values (GraphPad Prism 4.00, 2003). The experimental points of the time courses of H 2 O 2 curves were fitted with a one-phase exponential association equation. a, p < 0.05 compared to control (Student’s t -test).

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Knockdown, Diffusion-based Assay, Fluorescence, Control

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Representative immunofluorescence confocal microscopy images of colocalization of AQP8 and AQP11 with TOMM20 in HeLa cells. ( A , C ) Green labeling indicates the presence of TOMM20, and red labeling indicates the expression of AQP8 or AQP11 (Hoechst (blue) counterstained nuclei). Yellow labeling shows colocalization signal of AQP8 or AQP11 with TOMM20. Scale bar, 5 μm. ( B , D ) The graph, measured in the white line position in panels ( A , C ), shows the fluorescence signals of AQP8 or AQP11 and TOMM20 staining.

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Immunofluorescence, Confocal Microscopy, Labeling, Expressing, Fluorescence, Staining

Journal: Scientific Reports

Article Title: 2-Hydroxypropyl-gamma-cyclodextrin overcomes NPC1 deficiency by enhancing lysosome-ER association and autophagy

doi: 10.1038/s41598-020-65627-4

Figure Lengend Snippet: HPγCD promotes autophagy in NPC1 fibroblasts. NPC1 mutant cells treated with HPγCD or HPβCD (1 mM, 72 h) were analyzed for expression of autophagy marker proteins by Western blot and confocal microscopy. Cells were lysed and immunoblotted for Beclin-1, SQSTM1/p62, or LC3B ( a ). The blots are from different parts of the same gel and delineated with dividing lines. Western blot was analyzed using GAPDH as a loading control and the fold changes in protein expression levels were calculated using densitometry. Protein levels of Beclin-1 ( b ), SQSTM1/p62 ( c ), and LC3B ( d ) were significantly increased by HPγCD or HPβCD treatment. Co-localization of autophagosome and lysosome markers was analyzed by confocal microscopy ( e ). Association of LAMP-1 (lysosome marker, green) with LC3B (autophagosome marker, red) was enhanced in HPγCD or HPβCD treated cells. Data are mean ± S.E.M. of triplicates and a representative of three independent experiments. Symbols indicate the relative level of significance compared with the control (***P < 0.001). Scale bar = 50 µm.

Article Snippet: Primary antibodies: antibodies for LAMP1 (15665 S, 9091 S), GAPDH (5174 S), Beclin-1 (3495 S), SQTM (88588 S), and LC3B (3868 S) are from Cell Signaling Technology (Danvers, MA); antibodies for calnexin (PIPA534754), ORP2 (PA521891), ORP5 (PA518221), STARD3 (PA1562), VAPA (PA552660), Rab7 (PA5-52369), and RILP (PA534357) are from Thermo Fischer Scientific; antibodies for ORP1L (ab131165), calreticulin (ab92516), and TOMM20 (ab78547) are from Abcam (Cambridge, MA); antibodies for LAMP2 (sc-18822) and TFEB (sc-166736) are from Santa Cruz (Dallas, TX).

Techniques: Mutagenesis, Expressing, Marker, Western Blot, Confocal Microscopy, Control