Journal: bioRxiv

Article Title: Glucocorticoid-mediated Aβ and SCG10 upregulation evoke microtubule dysfunction and memory deficits

doi: 10.1101/322610

Figure Lengend Snippet: ( A ) The cells were incubated with cortisol (1 μM) for 2 h and then harvested. TOMM20 was co-immunoprecipitated with anti-IP3R and -Bcl-2 antibodies (the left side). Expression of IP3R, Bcl-2, TOMM20, and β-actin in total cell lysates is shown in the right side. Data are reported as a mean ± SE of four independent experiments. ** indicates p<0.01 versus control. ( B ) The cells were incubated with cortisol (1 μM) for various time and then harvested. GR was co-immunoprecipitated with anti-Bcl-2, -IP3R and -TOMM20 antibodies (the left side). Expression of Bcl-2, IP3R, TOMM20, GR, and β-actin in total cell lysates is shown in the right side. Data are reported as a mean ± SE of four independent experiments. *,** indicates p<0.05, p<0.01 versus control, respectively. ( C ) The cells were incubated with RU 486 (1 μM) for 30 min before cortisol treatment (1 μM) for 2 h. Co-localization of IP3R (green) and VDAC1 (red) was visualized with SRRF imaging system. DAPI was used for nuclear counterstaining (blue). Data are reported as a mean ± SE of five independent experiments. ** indicates p<0.01 versus control and ## indicates p<0.01 versus cortisol alone. Scale bars represent 20 μm (magnification, × 900). ( D ) Knockdown of bcl-2 was done using siRNA transfection for 24 h and then cells were treated with cortisol (1 μM) during 2 h. The cells underwent proximal ligation assay (PLA) and the red fluorescence indicates the co-localization between IP3R and VDAC1. DAPI was used for nuclear counterstaining (blue). Data are acquired by SRRF imaging system and reported as a mean ± SE of four independent experiments. ** indicates p<0.01 versus control and ## indicates p<0.01 versus cortisol alone. Scale bars represent 20 μm (magnification, × 900). ( E ) Knockdown of bcl-2 was done using siRNA transfection for 24 h and then cells were treated with cortisol (1 μM) during 2 h. mfn2 was co-immunoprecipitated with an anti-PACS2 antibody (the left side). Expression of PACS2, mfn2, and β-actin in total cell lysates is shown in the right side. Data are reported as a mean ± SE of four independent experiments. * indicates p<0.05 versus control and # indicates p<0.05 versus cortisol alone. ( F ) The hippocampus of mice exposed to vehicle or corticosterone (10 mg/kg) for 2 h was collected and lysed. VDAC1 was co-immunoprecipitated with anti-IP3R, -GR, and -Bcl-2 antibodies (the left side). Expression of IP3R, GR, Bcl-2, VDAC1, and β-actin in total cell lysates is shown in the right side. Data are reported as a mean ± SE of five independent experiments. *, ** indicates p<0.05, p<0.01 versus vehicle, respectively. ( G ) Slide samples for IHC of mice with vehicle or corticosterone (10 mg/kg) for 2 h underwent PLA and the red fluorescence indicates the co-localization between IP3R and VDAC1. DAPI was used for nuclear counterstaining (blue). Data are reported as a mean ± SE of five independent experiments. ** indicates p<0.01 versus vehicle. Scale bars, 200 μm (magnification, × 200). ( H ) The hippocampus of mice exposed to vehicle or corticosterone (10 mg/kg) for 2 h was collected and lysed. mfn2 was co-immunoprecipitated with an anti-PACS2 antibody (the left side). Expression of PACS2, mfn2, and β-actin in total cell lysates is shown in the right side. Data are reported as a mean ± SE of five independent experiments. ** indicates p<0.01 versus vehicle. ( I ) Slide samples for IHC of mice with vehicle or corticosterone (10 mg/kg) for 2 h were immunostained with PACS2 (green) and mfn2 (red). DAPI was used for nuclear counterstaining (blue). Scale bars, 200 μm (magnification, × 200). Data are reported as a mean ± SE of five independent experiments. ** indicates p<0.01 versus vehicle.

Article Snippet: The antibodies of COX IV, Hsp90, Hsp70, PACS2, PTPIP51, and the chemical cortisol-BSA were obtained from Cusabio (Wuhan, Hubei, China).

Techniques: Incubation, Immunoprecipitation, Expressing, Control, Imaging, Knockdown, Transfection, Ligation, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis

doi: 10.1007/s00018-022-04189-2

Figure Lengend Snippet: Pacs2 is decreased upon Chop overexpression. A MLE12 cells were either not induced or were induced with 1 µg/ml doxycycline for Chop expression for 12 and 24 h, followed by immunoblots for the given proteins from total lysates. B Quantification of significant decrease in Pacs2 protein levels upon Chop induction. Relative protein amounts were normalized to Gapdh and their level in −dox cells was set as one. C Representative immunoflourescence images of Pacs2 (green) in −dox or + dox-treated cells for 24 h. Nuclei were stained with DAPI (blue), scale bar = 60 µm. D Fluorescence intensity was quantified using ImageJ, and its intensity in −dox cells was set to one. Blots and analysis were performed from n = 3 experiments and statistical significance is indicated as: ** p ≤ 0.01

Article Snippet: Transient transfection was performed in a six-well dish using 4 μg PACS2-GFP (OriGene, Rockville, USA) or empty-GFP using 4 μl Lipofectamine ® 2000 Reagent (Invitrogen, Germany) per well or with non-targeting (NT) or Pacs2 siRNA (both from Santa Cruz) for 48 h as per manufacturer’s instructions.

Techniques: Over Expression, Expressing, Western Blot, Staining, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis

doi: 10.1007/s00018-022-04189-2

Figure Lengend Snippet: Pacs2 overexpression rescues phenotype of Chop-induced cells. MLE12 cells were either mock transfected, or transfected with empty-Myc or Myc-PACS2 for 4 h, followed by no dox (− dox) or with dox (+ dox, 12 h) treatment for Chop overexpression, followed by ( A ) proximity ligation assay with antibodies against calnexin and VDAC1 and subsequent fluorescence microscopy, scale bar = 60 µm. B Fluorescence intensity was quantified using ImageJ, its intensity in untreated −dox cells was set to one. C Immunoblots for the indicated proteins. D Relative protein levels of Pacs2 and E cleaved caspase 3 were normalized to Gapdh and their level in −dox cells was set as one. Representative images and stainings from n of 3 independent experiments are shown, statistical significance is indicated as: * p ≤ 0.05, *** p ≤ 0.001

Article Snippet: Transient transfection was performed in a six-well dish using 4 μg PACS2-GFP (OriGene, Rockville, USA) or empty-GFP using 4 μl Lipofectamine ® 2000 Reagent (Invitrogen, Germany) per well or with non-targeting (NT) or Pacs2 siRNA (both from Santa Cruz) for 48 h as per manufacturer’s instructions.

Techniques: Over Expression, Transfection, Proximity Ligation Assay, Fluorescence, Microscopy, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis

doi: 10.1007/s00018-022-04189-2

Figure Lengend Snippet: Modulation of Trpv1 improves ER–mitochondrial tethering in cells overexpressing Chop. A Immunoblot analysis of Trpv1 in total cell lysates of MLE12 cells untreated or treated with dox for Chop expression for 12 and 24 h. B Relative protein level of Trpv1 were normalized to Gapdh and its level in −dox cells was set as one. C MLE12 cells were either left untreated or treated with dox for 12 h, followed by treatments with DMSO or CPS at indicated concentrations followed by immunoblots for the indicated proteins. D and E Quantification of Trpv1 ( D ) and Pacs2 ( E ) protein expression is shown. Relative protein amounts were normalized to Gapdh and their mean value in respective DMSO-treated controls was set as one. F Representative fluorescence images following proximity ligation assay with antibodies against calnexin and VDAC1, and cells treated with CPS at the indicated concentrations or DMSO upon Chop induction are shown, scale bar = 60 µm. G Fluorescence intensity was quantified using ImageJ, its intensity in −dox, DMSO-treated cells was set to one. ‘ n ’ of three independent experiments were performed and statistical significance is indicated as: * p ≤ 0.05, *** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: Transient transfection was performed in a six-well dish using 4 μg PACS2-GFP (OriGene, Rockville, USA) or empty-GFP using 4 μl Lipofectamine ® 2000 Reagent (Invitrogen, Germany) per well or with non-targeting (NT) or Pacs2 siRNA (both from Santa Cruz) for 48 h as per manufacturer’s instructions.

Techniques: Western Blot, Expressing, Fluorescence, Proximity Ligation Assay

Journal: Cellular and Molecular Life Sciences

Article Title: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis

doi: 10.1007/s00018-022-04189-2

Figure Lengend Snippet: Modulating PACS2–TRPV1 axis with CPS rescues ER–mitochondrial tethering and PACS2 protein in cells overexpressing SPC Δexon4 . A MEL188 cells were stably transfected with either SPC WT or SPCΔ exon4 plasmids or left un-transfected, followed by immunoblots for the given proteins. B and C Quantification of PACS2 ( B ) and CHOP ( C ) protein levels are shown. Relative protein amounts were normalized to GAPDH and their mean value in untransfected cells was set as one. D Representative fluorescence microscopy images following proximity ligation assay with antibodies against calnexin and VDAC1 in SPC WT or SPCΔ exon4 overexpressing cells, scale bar = 60 µm. E Fluorescence intensity was quantified using ImageJ, and its intensity in untransfected cells was set to one. F Cells stably expressing SPC WT or SPCΔ exon4 or control cells were treated with DMSO or CPS, followed by immunoblotting of PACS2 and GAPDH. G Quantification of PACS2 protein expression is shown. Relative protein amounts were normalized to GAPDH and its mean value in control cells was set as one. H Fluorescence microscopy images following proximity ligation assay with antibodies against calnexin and VDAC1 in control cells or in cells overexpressing SPC WT or SPCΔ exon4 , followed by DMSO or CPS treatments at the indicated doses, scale bar = 60 µm. I Fluorescence intensity was quantified using ImageJ, and its intensity in untreated control cells was set as one. ‘ n ’ of three independent experiments were performed and statistical significance is indicated as: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: Transient transfection was performed in a six-well dish using 4 μg PACS2-GFP (OriGene, Rockville, USA) or empty-GFP using 4 μl Lipofectamine ® 2000 Reagent (Invitrogen, Germany) per well or with non-targeting (NT) or Pacs2 siRNA (both from Santa Cruz) for 48 h as per manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Western Blot, Fluorescence, Microscopy, Proximity Ligation Assay, Expressing, Control

Journal: Cellular and Molecular Life Sciences

Article Title: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis

doi: 10.1007/s00018-022-04189-2

Figure Lengend Snippet: PACS2 protein and ER–mitochondrial tethering are decreased in IPF AECII. A Representative immunofluorescence images for PACS2 (red) and ABCA3 (green) in IPF and Donor lung sections, and nuclei were stained with DAPI (blue) scale bar = 25 µm. B Fluorescence intensity of PACS2 was quantified using ImageJ, and its intensity in Donor sections was set as one. Lung sections from seven IPF to seven Donors were used for stainings. Statistical significance is indicated as: * p ≤ 0.05. C Representative transmission electron microscopic images from alveolar epithelial type II cells (characterized by the presence of lamellar bodies (LB)) are shown. At the ultrastructural level, the appearance of the contact zones between ER and mitochondria (Mito) in IPF differed from the healthy control. The closest contacts (arrows) appeared to be rather point shaped with the ER piston-like widened in IPF. In healthy controls, the contacts were in general elongated and more laminar so that the ER and mitochondrial membranes had a parallel run

Article Snippet: Transient transfection was performed in a six-well dish using 4 μg PACS2-GFP (OriGene, Rockville, USA) or empty-GFP using 4 μl Lipofectamine ® 2000 Reagent (Invitrogen, Germany) per well or with non-targeting (NT) or Pacs2 siRNA (both from Santa Cruz) for 48 h as per manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Fluorescence, Transmission Assay, Control

Journal: Cellular and Molecular Life Sciences

Article Title: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis

doi: 10.1007/s00018-022-04189-2

Figure Lengend Snippet: Modulation of TRPV1 decreases apoptosis and COLA1A level in PCLS of IPF patients. A PCLS from explanted IPF patient lungs were treated with either DMSO or CPS with the indicated concentrations for 8 h, followed by immunoblot analysis for PACS2, cleaved caspase 3 and GAPDH. B - D Quantification of PACS2 ( B ) and cl.caspase3 ( C ) protein levels is shown. Relative protein amounts were normalized to GAPDH and their mean value in DMSO-treated PCLS was set as one. D Immunofluorescence staining for cl.caspase 3 (red) in AECII (ABCA3, green) on IPF PCLS upon Veh, CPS or Nin treatment for 8 h. Nuclei were stained with DAPI (blue), scale bar = 60 µm. E Fluorescence intensity of cleaved caspase 3 in ABCA3-positive cells was quantified using ImageJ, and its intensity in DMSO-treated cells was set as one. F Immunofluorescence staining for COLA1A on IPF PCLS upon Veh, CPS or Nin treatment for 8 h. Nuclei were stained with DAPI (blue), scale bar = 60 µm. H Fluorescence intensity of COLA1A was quantified using ImageJ, and its intensity in DMSO-treated cells was set as one. Stainings and analysis were performed in PCLS performed from three IPF patients and significance is indicated as: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: Transient transfection was performed in a six-well dish using 4 μg PACS2-GFP (OriGene, Rockville, USA) or empty-GFP using 4 μl Lipofectamine ® 2000 Reagent (Invitrogen, Germany) per well or with non-targeting (NT) or Pacs2 siRNA (both from Santa Cruz) for 48 h as per manufacturer’s instructions.

Techniques: Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis

doi: 10.1007/s00018-022-04189-2

Figure Lengend Snippet: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis. Cartoon summarizing the results of this study: left side: upon ER stress/CHOP induction, SPC Δ exon4 overexpression or in IPF AECII where persistent pro-apoptotic ER stress is observed, PACS2 protein is severely decreased and, therefore, a reduction in ER–mitochondrial tethering is observed alongside with severe AECII apoptosis. Upon treatment with CPS, a TRPV1 modulator, PACS2 protein levels are restored, ER–mitochondrial tethering is rescued together with a decrease in AECII apoptosis and COL1A1

Article Snippet: Transient transfection was performed in a six-well dish using 4 μg PACS2-GFP (OriGene, Rockville, USA) or empty-GFP using 4 μl Lipofectamine ® 2000 Reagent (Invitrogen, Germany) per well or with non-targeting (NT) or Pacs2 siRNA (both from Santa Cruz) for 48 h as per manufacturer’s instructions.

Techniques: Over Expression

Journal: Cellular and Molecular Life Sciences

Article Title: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis

doi: 10.1007/s00018-022-04189-2

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: Transient transfection was performed in a six-well dish using 4 μg PACS2-GFP (OriGene, Rockville, USA) or empty-GFP using 4 μl Lipofectamine ® 2000 Reagent (Invitrogen, Germany) per well or with non-targeting (NT) or Pacs2 siRNA (both from Santa Cruz) for 48 h as per manufacturer’s instructions.

Techniques: Western Blot, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: AKT/PACS2 Participates in Renal Vascular Hyperpermeability by Regulating Endothelial Fatty Acid Oxidation in Diabetic Mice

doi: 10.3389/fphar.2022.876937

Figure Lengend Snippet: PACS2 increased expression in endothelial cells response to HGHF at the MAMs. (A). Western blot analysis of MAM-related proteins. ACTIN used as loading control. (B) . Quantitative analysis of the expression of FACL4, GRP75, and PACS2 in A. The band densities were quantitated and normalized to the corresponding ACTIN. (C) . Immunofluorescence imaging of cells stained for CD31 (red), PACS2 (green), and nucleus (DAPI, blue) in mouse kidney tissues. (D) . Transfection efficiency of PACS2 siRNA in HUVECs. (E) . Quantitative analysis of PACS2 knockdown in D. (F) . Confocal imaging of cells double-labeled with ER-Tracker Blue and Mito Tracker Deep Red in HUVECs. Single optical sections are shown and the purple ones indicate the co-localization of mitochondria and ER. Bar, 10 µm. (G) . Quantitative analysis of the co-localization ratio in F. Representative images are shown, or data are represented as the mean ± SD, n = 3. * p < 0.05.

Article Snippet: The following antibodies were used: anti-CPT1α (12252S, Cell Signaling Technology, Beverly, MA, USA), PACS2 (GTX17244, GeneTex, Irvine, CA, USA), VE-cadherin (ab33168, Abcam), β‐actin (A5441, Sigma‐Aldrich), AKT (2938S, Cell Signaling Technology), p-AKT (9018S, Cell Signaling Technology), Smad2 (5339T, Cell Signaling Technology), p-Smad2 (ab280888, Abcam, UK), PPARα (ab215270, Abcam, UK), and HRP conjugated goat anti-rabbit IgG secondary antibody (AS09 602, Agrisera, Vannas, Sweden).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Imaging, Staining, Transfection, Knockdown, Labeling

Journal: Frontiers in Pharmacology

Article Title: AKT/PACS2 Participates in Renal Vascular Hyperpermeability by Regulating Endothelial Fatty Acid Oxidation in Diabetic Mice

doi: 10.3389/fphar.2022.876937

Figure Lengend Snippet: Loss of PACS2 expression improves kidney barrier function and anti-fibrosis induced by HGHF. (A) . Immunofluorescence of kidney tissues. Bar, 50 μm (B) . Biochemical analysis of serum parameters in wild type (WT) and PACS2 −/− mice fed with or without HFD. (C) . Masson staining of kidney tissues, Magnification, ×100. (D) . Histogram of CVF in B. (E) . Transmission electron microscope imaging of mouse kidney ultrastructure. Magnification, ×15,000. Red M, mitochondria of renal endothelial cells; blue arrow, MAMs. Images of Evans blue staining of mouse kidneys. (F) . Photographs of Evans blue (EB)-stained mouse kidneys. (G) . Histogram of EB extravasation index. Representative images are shown or data are represented as the mean ± SD, n ≥ 5. * p < 0.05.

Article Snippet: The following antibodies were used: anti-CPT1α (12252S, Cell Signaling Technology, Beverly, MA, USA), PACS2 (GTX17244, GeneTex, Irvine, CA, USA), VE-cadherin (ab33168, Abcam), β‐actin (A5441, Sigma‐Aldrich), AKT (2938S, Cell Signaling Technology), p-AKT (9018S, Cell Signaling Technology), Smad2 (5339T, Cell Signaling Technology), p-Smad2 (ab280888, Abcam, UK), PPARα (ab215270, Abcam, UK), and HRP conjugated goat anti-rabbit IgG secondary antibody (AS09 602, Agrisera, Vannas, Sweden).

Techniques: Expressing, Immunofluorescence, Staining, Transmission Assay, Microscopy, Imaging

Journal: Frontiers in Pharmacology

Article Title: AKT/PACS2 Participates in Renal Vascular Hyperpermeability by Regulating Endothelial Fatty Acid Oxidation in Diabetic Mice

doi: 10.3389/fphar.2022.876937

Figure Lengend Snippet: PACS2 regulates barrier injury and p-Smad2 under HGHF blocked by inhibition of the p-AKT pathway in HUVECs. (A) . Western blot analysis of FN, p-smad2 and p-AKT expression under HGHF. (B) . Quantitation and normalization of FN, p-smad2 and p-AKT expression in A. (C) . Western blot analysis of PACS2, FN and p-smad2 and p-AKT expression under p-AKT inhibitor MK2206 and siPACS2. (D) . Quantitation and normalization of proteins expression in C.

Article Snippet: The following antibodies were used: anti-CPT1α (12252S, Cell Signaling Technology, Beverly, MA, USA), PACS2 (GTX17244, GeneTex, Irvine, CA, USA), VE-cadherin (ab33168, Abcam), β‐actin (A5441, Sigma‐Aldrich), AKT (2938S, Cell Signaling Technology), p-AKT (9018S, Cell Signaling Technology), Smad2 (5339T, Cell Signaling Technology), p-Smad2 (ab280888, Abcam, UK), PPARα (ab215270, Abcam, UK), and HRP conjugated goat anti-rabbit IgG secondary antibody (AS09 602, Agrisera, Vannas, Sweden).

Techniques: Inhibition, Western Blot, Expressing, Quantitation Assay

Journal: Frontiers in Pharmacology

Article Title: AKT/PACS2 Participates in Renal Vascular Hyperpermeability by Regulating Endothelial Fatty Acid Oxidation in Diabetic Mice

doi: 10.3389/fphar.2022.876937

Figure Lengend Snippet: Silencing PACS2 improves free fatty metabolism in HUVECs treated with HGHF. (A) . KEGG analysis of metabolomics in HUVECs under HGHF. (B) . Palmitic acid (PA)-based oxygen consumption rate (OCR) was measured in cells transfected with negative siRNA (NeSiRNA) or PACS2 siRNA (siPACS2) and treated with or without HGHF. (C) . Determination of cellular NADPH/NADP ratio. (D) . Determination of cellular ATP levels. (E) . Western blot analysis of PACS2 and carnitine palmitoyl-transferase 1α (CPT1α) expression. (F) . Quantitation and normalization of PACS2 expression in E. (G) . Quantitation and normalization of CPT1α expression in E. (H) . Immunohistochemical staining of CPT1α in mouse kidney. Magnification, ×200. (I) . Oil Red staining of mouse kidney. Magnification, ×200. Representative images are shown or data are represented as the mean ± SD, n = 3. * p < 0.05.

Article Snippet: The following antibodies were used: anti-CPT1α (12252S, Cell Signaling Technology, Beverly, MA, USA), PACS2 (GTX17244, GeneTex, Irvine, CA, USA), VE-cadherin (ab33168, Abcam), β‐actin (A5441, Sigma‐Aldrich), AKT (2938S, Cell Signaling Technology), p-AKT (9018S, Cell Signaling Technology), Smad2 (5339T, Cell Signaling Technology), p-Smad2 (ab280888, Abcam, UK), PPARα (ab215270, Abcam, UK), and HRP conjugated goat anti-rabbit IgG secondary antibody (AS09 602, Agrisera, Vannas, Sweden).

Techniques: Transfection, Western Blot, Expressing, Quantitation Assay, Immunohistochemical staining, Staining

Journal: Frontiers in Pharmacology

Article Title: AKT/PACS2 Participates in Renal Vascular Hyperpermeability by Regulating Endothelial Fatty Acid Oxidation in Diabetic Mice

doi: 10.3389/fphar.2022.876937

Figure Lengend Snippet: Inhibition of fatty acid β-oxidation disturbs the barrier function of endothelial cells and eliminates the protective effect of PACS2. (A) . Immunofluorescence staining of VE-cadherin (green) in cells transfected with NeSiRNA or siPACS2 and treated with the fatty acid β-oxidation inhibitor etomoxir (ETO) or phosphate buffered saline (PBS) as vehicle control. Blue, DAPI. Bar, 20 µm. (B). Quantification of FITC-dextran leakage ratio of HUVECs within 15 min. Representative images are shown or data are represented as the mean ± SD, n = 3. * p < 0.05.

Article Snippet: The following antibodies were used: anti-CPT1α (12252S, Cell Signaling Technology, Beverly, MA, USA), PACS2 (GTX17244, GeneTex, Irvine, CA, USA), VE-cadherin (ab33168, Abcam), β‐actin (A5441, Sigma‐Aldrich), AKT (2938S, Cell Signaling Technology), p-AKT (9018S, Cell Signaling Technology), Smad2 (5339T, Cell Signaling Technology), p-Smad2 (ab280888, Abcam, UK), PPARα (ab215270, Abcam, UK), and HRP conjugated goat anti-rabbit IgG secondary antibody (AS09 602, Agrisera, Vannas, Sweden).

Techniques: Inhibition, Immunofluorescence, Staining, Transfection, Saline, Control

Journal: Frontiers in Pharmacology

Article Title: AKT/PACS2 Participates in Renal Vascular Hyperpermeability by Regulating Endothelial Fatty Acid Oxidation in Diabetic Mice

doi: 10.3389/fphar.2022.876937

Figure Lengend Snippet: A flow chart model proposal to explain the mechanism of renal vascular endothelial cell injury in DN. PACS2 is a critical component of the mechanism behind DN caused by HGHF. Under high glucose/high fat conditions, PACS2 is upregulated and increases the number of MAMs, followed by a decrease in CPT1α expression, free fatty (FA) β-oxidation, and NADPH production in renal vascular endothelial cells. All these metabolic changes will promote the internalization of VE-cadherin, disturb the barrier function of endothelial cells, and fibrosis increased ultimately lead to pathogenesis and development of DN.

Article Snippet: The following antibodies were used: anti-CPT1α (12252S, Cell Signaling Technology, Beverly, MA, USA), PACS2 (GTX17244, GeneTex, Irvine, CA, USA), VE-cadherin (ab33168, Abcam), β‐actin (A5441, Sigma‐Aldrich), AKT (2938S, Cell Signaling Technology), p-AKT (9018S, Cell Signaling Technology), Smad2 (5339T, Cell Signaling Technology), p-Smad2 (ab280888, Abcam, UK), PPARα (ab215270, Abcam, UK), and HRP conjugated goat anti-rabbit IgG secondary antibody (AS09 602, Agrisera, Vannas, Sweden).

Techniques: Expressing

Journal: Molecular Medicine

Article Title: PACS-2 deficiency in tubular cells aggravates lipid-related kidney injury in diabetic kidney disease

doi: 10.1186/s10020-022-00545-x

Figure Lengend Snippet: Tubule-specific Pacs-2 deletion aggravates kidney injury in HFD/STZ treated mice. A and B Western blot and quantification of PACS-2 expression showing that PACS-2 was knocked out in renal cortex. C Body weight and D blood glucose are shown. E Kidney weight/body weight (KW/BW) ratio, F Urine albumin-to-creatinine ratio (UACR) and urinary NAG in mice are shown. G Morphological examinations of tubular and glomerular changes by HE, PAS, Masson staining. Scale bars: 50 μm. H and I Quantification of tubular damage score and glomerular damage score of the kidneys in each group. All data are presented as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. n = 6

Article Snippet: PACS-2 siRNA was purchased from RiboBio Co., Ltd (China).

Techniques: Western Blot, Expressing, Staining

Journal: Molecular Medicine

Article Title: PACS-2 deficiency in tubular cells aggravates lipid-related kidney injury in diabetic kidney disease

doi: 10.1186/s10020-022-00545-x

Figure Lengend Snippet: Pacs-2 gene deficiency contributes to lipid accumulation in tubular cells of diabetic mice. A Representative images showing lipid deposition by immunohistochemistry of Adipophilin and Oil Red O staining of kidney from mice. Scale bars: 50 μm. B and C Relative Adipophilin expression and Oil Red O staining area. D Measurement of the mRNA expression of selected markers. E and F Western blot and quantification of FN and α-SMA. All data are presented as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. n = 6

Article Snippet: PACS-2 siRNA was purchased from RiboBio Co., Ltd (China).

Techniques: Immunohistochemistry, Staining, Expressing, Western Blot

Journal: Molecular Medicine

Article Title: PACS-2 deficiency in tubular cells aggravates lipid-related kidney injury in diabetic kidney disease

doi: 10.1186/s10020-022-00545-x

Figure Lengend Snippet: Overexpression of PACS-2 ameliorates lipid accumulation in HK-2 cells induced by HGPA. A Gene overexpression efficiency of PACS-2 by western blot analysis in PACS-2 plasmid transfected HK-2 cells. B Representative Oil Red O staining images of HK-2 cells in different groups. Scale bar: 20 μm. C Oil Red O staining area. D Cholesteryl ester content. E Free cholesterol content. F Total cholesterol content. G Free fatty acid content. All data are presented as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. n = 4

Article Snippet: PACS-2 siRNA was purchased from RiboBio Co., Ltd (China).

Techniques: Over Expression, Western Blot, Plasmid Preparation, Transfection, Staining

Journal: Molecular Medicine

Article Title: PACS-2 deficiency in tubular cells aggravates lipid-related kidney injury in diabetic kidney disease

doi: 10.1186/s10020-022-00545-x

Figure Lengend Snippet: Identification of Soat1 as a downstream molecule of Pacs-2. A Volcano plot of DEGs in kidney cortex of control Pacs-2 fl/fl mice and control PT-Pacs-2 −/− mice. Splashes represent different genes, and the gray splashes mean genes without significant different expression. The red splashes mean significantly up-regulated genes in control PT-Pacs-2 −/− mice, and the blue splashes mean significantly down-regulated genes in control PT-Pacs-2 −/− mice. B Heat map of top eight DEGs involved in lipid metabolism; n = 3 mice per group. C Real-time qRT-PCR showing the relative mRNA expression of Soat1 . n = 6 mice per group. D Representative immunofluorescence images of SOAT1 (green) in kidney tissues from each group. The nuclei were delineated by DAPI (blue). n = 6 mice per group. Bars = 50 μm. E Semiquantification analysis of protein expression. F Representative western blot bands of SOAT1, n-SREBP1, n-SREBP2 expression in the kidney cortex, n = 6 mice per group. G Relative band density. All data are presented as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: PACS-2 siRNA was purchased from RiboBio Co., Ltd (China).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Western Blot

Journal: Molecular Medicine

Article Title: PACS-2 deficiency in tubular cells aggravates lipid-related kidney injury in diabetic kidney disease

doi: 10.1186/s10020-022-00545-x

Figure Lengend Snippet: Treatment of HK-2 cells with PACS-2 and SOAT1 siRNA regulates lipid metabolism. A and B Representative western blot and quantification of PACS-2, SOAT1, n-SREBP1, n-SREBP2 in HK-2 cells. C Measurement of the mRNA expression of selected markers. D Representative images of cells stained with Bodipy (green); nuclei stained with Hoechst (blue). E Quantification of the Bodipy staining. F Cholesteryl ester content. G Free cholesterol content. H Total cholesterol content. I Free fatty acid content. All data are presented as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. n = 4

Article Snippet: PACS-2 siRNA was purchased from RiboBio Co., Ltd (China).

Techniques: Western Blot, Expressing, Staining