Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: NIC1 signaling regulates ER calcium levels. Schematic (I) Timeline of experiments assessing changes in ER calcium levels. Schematic (II) Timeline of experiments, in cells treated with siRNA before assessing changes in ER calcium levels. (A) Fluo4 fluorescence in HEK cells expressing NIC1-RFP ( ) or RFP ( ), at time-t (F t ) relative to onset (F 0 ) measured in calcium free medium at baseline and in response to 2 μM TG. (B) YFP/CFP ratio in HEK cells co-transfected with D1ER and NIC1-RFP ( ) or, Bcl-xL-RFP ( ) or, RFP ( ) and cultured for 36 h before imaging. (C) Schematic (not to scale) depicting - IP3R3, Grp75, VDAC1 and MCU- in the context of ER and mitochondria. (D–F) Ratio of YFP/CFP fluorescence in HEK cells, imaged 36 h after co-transfection with D1ER and NIC1-RFP ( ) or RFP ( ), in cells pre-treated with siRNA to IP3R3 (D-ii) or VDAC1 (E-ii) or MCU (F-ii) or scrambled control ( D-F-i , NIC1-RFP ( ) and RFP ( ). (D–F iii) Percent mRNA levels of the genes as shown in panels, in cells treated with siRNA to IP3R3 ( D , N=3) or VDAC1 ( E , N=2) or MCU ( F , N=3) and scrambled control. Data plotted as mean ± SD of the indicated number of cells in 6-12 fields across three (A, D, F) or two (B, D) independent experiments. *** indicates significant difference at all time points with p-values ≤0.001 and ns: not significant, examined using unpaired student’s t-test.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques: Fluorescence, Expressing, Transfection, Cell Culture, Imaging, Cotransfection, Control

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: NIC1-mediated Treg survival requires IP3Rs, Grp75 and MCU. (A) Percent apoptotic nuclei in activated WT Tregs cultured without IL-2 for 24 h with vehicle control (UT) or, 5 μM Xestospongin C or, 10 μM MKT-077 or, 10 μM Ru360. (B, C) Percent apoptotic nuclei in Notch1 -/- Tregs transduced with pBABE or NIC1-NES (B) or pMIG or Bcl-xL (C), and cultured without IL-2 for 24 h with vehicle control, or 5 μM Xestospongin C or, 10 μM MKT-077 or, 10 μM Ru360. (D) Basal and maximum OCR, computed as described in methods, in Notch1 -/- Tregs transduced with pBABE or NIC1-NES. (E) Immunoblot of whole cell lysates prepared from activated WT Tregs cells cultured with or without 10 μM GSI-X or 10 μM MKT-077 for 8 h and the samples run in duplicate. Membrane was probed either for (p)PDH, Notch1 and Tubulin (E-i) or PDH, Notch1 and Tubulin (E-ii) . The immunoblot is representative of two independent experiments. Data are mean ± SD of three independent experiments (A–C) and readings in 4 wells from two independent experiments (D). ** indicates significant difference with p-value ≤0.01 and ns, not significant, examined using unpaired student’s t-test.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques: Cell Culture, Control, Transduction, Western Blot, Membrane

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: Notch1 activity regulates Grp75 protein levels. (A) Immunoblots of whole cell lysates prepared from activated Notch1 +/+ (Cre-ve) and Notch1 -/- (Cre+ve; Cd4-Cre::Notch1 lox/lox ) Tregs, run in duplicate. Membranes were sequentially probed for Grp75, VADC1 (*VDAC1 band), and Actin (A-i) or Notch1, MCU and Actin (A-ii). Mean ± SD values below are the densitometry analysis of Grp75, VDAC1 and MCU relative to Actin. (B) Representative Z-projected confocal images of Notch1 +/+ and Notch1 -/- Tregs immune-stained with an antibody to Grp75 (green). scale bar: 5 μm. Images are representative of 106 Notch1 +/+ Tregs and 82 Notch1 -/- Tregs. (C) Relative transcript levels of indicated genes in Notch1 +/+ and Notch1 -/- activated Tregs. (D) Immunoblots of cell lysates from Notch1 -/- Tregs transduced with pBABE or NIC1-NES probed for Notch1, Grp75 and Actin. Mean ± SD values below are the densitometry analysis of Grp75 relative to Actin. (E) Percent apoptotic nuclei in Notch1 -/- Tregs transduced with pBABE or Grp75, cultured without IL-2 for 24 h with 5μM Xestospongin C or, 10 μM MKT-077 or, 10 μM Ru360. (F) Basal and maximum OCR in Notch1 -/- Tregs transduced with pBABE or Grp75. Control (pBABE) condition in panels E and F are common Figures 5B, D as these were tested in the same experiment, as described in methods . (G, H) Cell lysates of WT activated Tregs cultured for 6 h without IL-2 were subject to immunoprecipitation using an antibody to Notch1 (G) or Grp75 (H) or, IgG (Isotype control), and associated proteins analyzed by western blotting for Grp75, VDAC1 (*shows VDAC1), Notch1, Vps34, Actin and IgG (Isotype control). Immunoblots are representative of three independent experiments (A) or, two independent experiments (D, G, H) . Data show the mean ± SD of three independent experiments (C, E) and readings in 4 wells from two independent experiments (F). * and ** indicates significant difference with p-value ≤0.05 and ≤0.01 respectively, and ns: not significant, examined using unpaired student’s t-test.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques: Activity Assay, Western Blot, Staining, Transduction, Cell Culture, Control, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: Summary of key outcomes. Non-nuclear localized NIC1 interacts with Grp75 and VDAC1 in a complex that modulates calcium homeostasis in mitochondria with consequences to cellular metabolism and survival. The schematic is not to scale.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques:

Journal: Journal of the American Heart Association

Article Title: S100a8/9 (S100 Calcium Binding Protein a8/9) Promotes Cardiac Hypertrophy Via Upregulation of FGF23 (Fibroblast Growth Factor 23) in Mice

doi: 10.1161/jaha.122.028006

Figure Lengend Snippet: Figure 5. S100a8/9 activated calcineurin/NFAT signaling pathway during phenylephrine-induced hypertrophy of cardiomyocytes in vitro. A, Cell areas of NRCMs treated with several inhibitors of hypertrophy-related kinases (n=5). cyclosporin A (an inhibitor of calcineurin), VIVIT (an inhibitor of NFAT), CCT129957 (an inhibitor of PLCγ), PD169316 (an inhibitor of p38), SP600125 (an inhibitor of JNK), U0126 (an inhibitor of ERK), BMS-345541 (an inhibitor of NF-κB), and AKTi (an inhibitor of AKT) were used. B, mRNA levels of atrial natriuretic peptide (ANP) in indicated groups (n=5). C and D, Calcineurin activity in the hearts following AB with overexpression or knockdown of S100a9 (n=6). E and F, NFAT transcriptional activity in the hearts following AB with overexpression or knockdown of S100a9 (n=6). G and H, mRNA level of calcineurin 1 (RCAN1) in the hearts following AB with overexpression or knockdown of S100a9 (n=6). I, Representative Western blots and the statistical results of the protein levels (n=6). Values represent the mean±SEM. *P<0.05 vs the matched control group. AB indicates aortic banding; ANP, atrial natriuretic peptide; NF-κB, nuclear factor-κB; NFAT, nuclear factor of activated T cells; NRCM, neonatal rat cardiomyocyte; PCNA, proliferating cell nuclear antigen; PLCγ, phospholipase C-γ; P-PLCγ, phospho-PLCγ; RCAN1, regulator of calcineurin 1; and S100a8/9, S100 calcium binding protein a8/9.

Article Snippet: The inhibitors, PD169316, SP600125, U0126, VIVIT, AKTi, CCT129957, and cyclosporin A were purchased from MedChemExpress (Wuhan, China).

Techniques: In Vitro, Activity Assay, Over Expression, Knockdown, Western Blot, Control, Binding Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Insufficient Dose of ERCC8 Protein Caused by a Frameshift Mutation Is Associated With Keratoconus With Congenital Cataracts

doi: 10.1167/iovs.63.13.1

Figure Lengend Snippet: Pedigree and genetic mutation of the family with keratoconus and congenital cataracts. ( A ) Pedigree. ( B ) Sanger sequencing confirms the c.394-398del (p. L132Nfs*6) mutation identified by WES (I-2 and II-1) and HRM (KC5). ( C ) Cross-species comparison of the region of ERCC8 indicates that the identified mutation affects highly conserved residues. ( D ) Domain analysis of the p. L132Nfs*6 mutation. ( E ) The 3D structures of mutant and wild type protein.

Article Snippet: Primary antibodies included those of collagen IV (ab6586; Abcam), ERCC8 (sc-376981; Santa Cruz), SOD1 (MABC684; Millipore), γ-H2AX (AP0099; Abclonal), NF-kB p65 (phosphor S536, ab76302; Abcam), MMP1 (ab52631; Abcam), Caspase 3 (D320074; Sangon), and GAPDH (KC-5G5; Kangchen, Shanghai, China).

Techniques: Mutagenesis, Sequencing, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: Insufficient Dose of ERCC8 Protein Caused by a Frameshift Mutation Is Associated With Keratoconus With Congenital Cataracts

doi: 10.1167/iovs.63.13.1

Figure Lengend Snippet: Pathogenicity Assessment of ERCC8 Mutation

Article Snippet: Primary antibodies included those of collagen IV (ab6586; Abcam), ERCC8 (sc-376981; Santa Cruz), SOD1 (MABC684; Millipore), γ-H2AX (AP0099; Abclonal), NF-kB p65 (phosphor S536, ab76302; Abcam), MMP1 (ab52631; Abcam), Caspase 3 (D320074; Sangon), and GAPDH (KC-5G5; Kangchen, Shanghai, China).

Techniques: Mutagenesis, Sequencing, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Insufficient Dose of ERCC8 Protein Caused by a Frameshift Mutation Is Associated With Keratoconus With Congenital Cataracts

doi: 10.1167/iovs.63.13.1

Figure Lengend Snippet: Expression of ERCC8 in different mouse tissues. ( A ) Representative figures of immunofluorescence staining of ERCC8 in the mouse eye (the upper was a panoramic image, and the lower were local images). ( B ) The mRNA expression levels of ERCC8 in different mouse tissues.

Article Snippet: Primary antibodies included those of collagen IV (ab6586; Abcam), ERCC8 (sc-376981; Santa Cruz), SOD1 (MABC684; Millipore), γ-H2AX (AP0099; Abclonal), NF-kB p65 (phosphor S536, ab76302; Abcam), MMP1 (ab52631; Abcam), Caspase 3 (D320074; Sangon), and GAPDH (KC-5G5; Kangchen, Shanghai, China).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Insufficient Dose of ERCC8 Protein Caused by a Frameshift Mutation Is Associated With Keratoconus With Congenital Cataracts

doi: 10.1167/iovs.63.13.1

Figure Lengend Snippet: Mutant ERCC8 constructs and expression in human embryonic kidney 293 cells. ( A ) The fluorescence intensity of GFP after transfection for 48 hours. ( B ) mRNA expression levels of ERCC8 and TCR-related genes ( CSB , XPA , RPA , and XPG ) in overexpressed wild type and mutant cells. ( C ) Western blot results of ERCC8 protein in overexpressed wild type and mutant cells. ( D ) Statistical results of the Western blot; N = 3, *, P < 0.05.

Article Snippet: Primary antibodies included those of collagen IV (ab6586; Abcam), ERCC8 (sc-376981; Santa Cruz), SOD1 (MABC684; Millipore), γ-H2AX (AP0099; Abclonal), NF-kB p65 (phosphor S536, ab76302; Abcam), MMP1 (ab52631; Abcam), Caspase 3 (D320074; Sangon), and GAPDH (KC-5G5; Kangchen, Shanghai, China).

Techniques: Mutagenesis, Construct, Expressing, Fluorescence, Transfection, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Insufficient Dose of ERCC8 Protein Caused by a Frameshift Mutation Is Associated With Keratoconus With Congenital Cataracts

doi: 10.1167/iovs.63.13.1

Figure Lengend Snippet: Knockdown of ERCC8 in human corneal fibroblast cells. ( A ) The mRNA expression levels of ERCC8 after being transfected by ERCC8 siRNA. ( B ) The mRNA expression levels of TCR-related genes ( CSB , XPA , RPA , and XPG ) after being transfected by ERCC8 siRNA. ( C ) Western blot results of ERCC8, collagen Ⅳ, and SOD1 proteins after being transfected by ERCC8 siRNA. ( D ) Statistical results of the Western blot; N = 3, *, P < 0.05.

Article Snippet: Primary antibodies included those of collagen IV (ab6586; Abcam), ERCC8 (sc-376981; Santa Cruz), SOD1 (MABC684; Millipore), γ-H2AX (AP0099; Abclonal), NF-kB p65 (phosphor S536, ab76302; Abcam), MMP1 (ab52631; Abcam), Caspase 3 (D320074; Sangon), and GAPDH (KC-5G5; Kangchen, Shanghai, China).

Techniques: Knockdown, Expressing, Transfection, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Insufficient Dose of ERCC8 Protein Caused by a Frameshift Mutation Is Associated With Keratoconus With Congenital Cataracts

doi: 10.1167/iovs.63.13.1

Figure Lengend Snippet: Cell viability and DNA damage assays of HTK treated by H 2 O 2 after ERCC8 siRNA transfection. ( A ) Cell viability assay results. ( B ) Hoechst staining results. ( C ) Western blot results of ERCC8, γ- H2AX, and phospho p65 protein. ( D ) Statistical results of the Western blot; N = 3, *, P < 0.05.

Article Snippet: Primary antibodies included those of collagen IV (ab6586; Abcam), ERCC8 (sc-376981; Santa Cruz), SOD1 (MABC684; Millipore), γ-H2AX (AP0099; Abclonal), NF-kB p65 (phosphor S536, ab76302; Abcam), MMP1 (ab52631; Abcam), Caspase 3 (D320074; Sangon), and GAPDH (KC-5G5; Kangchen, Shanghai, China).

Techniques: Transfection, Viability Assay, Staining, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Insufficient Dose of ERCC8 Protein Caused by a Frameshift Mutation Is Associated With Keratoconus With Congenital Cataracts

doi: 10.1167/iovs.63.13.1

Figure Lengend Snippet: Cell viability, apoptosis, and DNA damage assays of HLECs treated by H 2 O 2 after ERCC8 siRNA transfection. ( A ) Cell viability assay results. ( B ) The mRNA expression levels of TCR-related genes. ( C ) Representative figures of immunofluorescence staining of cell apoptosis. ( D ) Statistical results of the cell apoptosis analysis. ( E ) The mRNA expression levels of UPR-related genes. ( F ) Western blot results of SOD1, γ- H2AX, and caspase 3 protein. ( D ) Statistical results of the Western blot; N = 3, *, P < 0.05.

Article Snippet: Primary antibodies included those of collagen IV (ab6586; Abcam), ERCC8 (sc-376981; Santa Cruz), SOD1 (MABC684; Millipore), γ-H2AX (AP0099; Abclonal), NF-kB p65 (phosphor S536, ab76302; Abcam), MMP1 (ab52631; Abcam), Caspase 3 (D320074; Sangon), and GAPDH (KC-5G5; Kangchen, Shanghai, China).

Techniques: Transfection, Viability Assay, Expressing, Immunofluorescence, Staining, Western Blot