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: Scientific Reports

Article Title: DGCR8 regulates multiple processes of transcription coupled nucleotide excision repair

doi: 10.1038/s41598-026-38338-5

Figure Lengend Snippet: S153A mutation disrupts UV-induced interactions between DGCR8 and TC-NER factors in MEFs. (a, b) Quantification and representative PLA images showing CSB–CSA ( a ) and DGCR8–CSB ( b ) interactions in MEFs. Wild-type, heterozygous (wt/S153A), and homozygous (S153A/S153A) MEFs were irradiated with 10 J/m 2 UV-C (purple dots) or left untreated (gray dots), followed by 2 hours of recovery. PLA signals (red) were detected in nuclei counterstained with DAPI (blue). Horizontal black bars indicate the median of each group. Asterisks denote significant differences relative to non-irradiated or between genotypes (*** p < 0.001). White scale bars, 10 μm.

Article Snippet: The following commercial antibodies were also used: DGCR8 (10996-1-AP, Proteintech; sc-377249, Santa Cruz), RNA polymerase II (CDT-pS5, sc-55492[F-12], Santa Cruz), CSB (sc-25370, Santa Cruz), CSA (sc-376981, Santa Cruz), USP7 (300 − 033, Bethyl), UVSSA (GT816, GeneTex), CHK1-pS345 (2341 L, CST), ATM-pS1981 (4526 S, CST), SPT16 (sc-165987, Santa Cruz), SMARCA5 (sc-365727, Santa Cruz), CPD (CAC-NM-DND-001, Cosmo Bio), 6-4PP (CAC-NM-DND-002, Cosmo Bio), and GFP (50430-2-AP, Proteintech; AE012, Abclonal).

Techniques: Mutagenesis, Irradiation

Journal: Cells

Article Title: A Novel Missense Mutation in ERCC8 Co-Segregates with Cerebellar Ataxia in a Consanguineous Pakistani Family

doi: 10.3390/cells11193090

Figure Lengend Snippet: Pedigree, genotypes, and MRIs of the consanguineous family segregating the ERCC8 variant. ( A ) Pedigree and genotypes of the family segregating ERCC8 mutation c.176T>C. Squares and circles indicate males and females, respectively. The symbols in black filled represent the affected members. The symbol with a diagonal line indicates a deceased individual. The numbers to the left of pedigree show generation number while those above the symbols denote individuals within that generation. ( B ) T2-weighted brain MRI of the normal brother (IV:5) shows a healthy cerebellum. Patient (IV:3) has prominent cerebellar atrophy/degeneration (upper panel) and prominent intra and extra cerebral CSF spaces (lower panel) shown on axial section of T2-weighted image. Affected brother (IV:2) has prominent cerebellar atrophy/degeneration (upper panel) and mildly dilated intra and extra cerebral CSF spaces (lower panel) shown on axial section of T2-weighted image. Affected brother (IV:1) has very mild atrophy/degeneration of cerebellar folia (upper panel) on axial section of T2-weighted image.

Article Snippet: Overexpression vectors containing WT ERCC8 transcript variant 1 cDNA were purchased (Origene, Rockville, MD, USA; Cat# RC214392) and site-directed mutagenesis was performed (Agilent QuikChange II XL; Cat# 200521; Agilent Technologies, Santa Clara, CA, USA) to generate mutant ERCC8 expression vectors using following primers: forward 5′-TCTGAACCACCTGATAACGTGTATCTCCCTTCAACAG-3′ and reverse 5′-CTGTTGAAGGGAGATACACGTTATCAGGTGGTTCAGA-3′.

Techniques: Variant Assay, Mutagenesis

Journal: Cells

Article Title: A Novel Missense Mutation in ERCC8 Co-Segregates with Cerebellar Ataxia in a Consanguineous Pakistani Family

doi: 10.3390/cells11193090

Figure Lengend Snippet: Clinical manifestations of patients with autosomal recessive ERCC8 mutation.

Article Snippet: Overexpression vectors containing WT ERCC8 transcript variant 1 cDNA were purchased (Origene, Rockville, MD, USA; Cat# RC214392) and site-directed mutagenesis was performed (Agilent QuikChange II XL; Cat# 200521; Agilent Technologies, Santa Clara, CA, USA) to generate mutant ERCC8 expression vectors using following primers: forward 5′-TCTGAACCACCTGATAACGTGTATCTCCCTTCAACAG-3′ and reverse 5′-CTGTTGAAGGGAGATACACGTTATCAGGTGGTTCAGA-3′.

Techniques: Mutagenesis

Journal: Cells

Article Title: A Novel Missense Mutation in ERCC8 Co-Segregates with Cerebellar Ataxia in a Consanguineous Pakistani Family

doi: 10.3390/cells11193090

Figure Lengend Snippet: List of autosomal recessive homozygous candidate variants derived from exome sequencing data. All variants except one in ERCC8 have been excluded due to no segregation and lack of evidence for brain related function/phenotype.

Article Snippet: Overexpression vectors containing WT ERCC8 transcript variant 1 cDNA were purchased (Origene, Rockville, MD, USA; Cat# RC214392) and site-directed mutagenesis was performed (Agilent QuikChange II XL; Cat# 200521; Agilent Technologies, Santa Clara, CA, USA) to generate mutant ERCC8 expression vectors using following primers: forward 5′-TCTGAACCACCTGATAACGTGTATCTCCCTTCAACAG-3′ and reverse 5′-CTGTTGAAGGGAGATACACGTTATCAGGTGGTTCAGA-3′.

Techniques: Derivative Assay, Sequencing, Mutagenesis, Variant Assay

Journal: Cells

Article Title: A Novel Missense Mutation in ERCC8 Co-Segregates with Cerebellar Ataxia in a Consanguineous Pakistani Family

doi: 10.3390/cells11193090

Figure Lengend Snippet: Analysis of the ERCC8 M59T variant and its 3D structure modeling. ( A ) Chromatograms of DNA sequence of healthy carrier IV:5 (top) indicates heterozygous for the variant, and affected individual IV:1 (bottom) indicates a homozygous missense mutation in ERCC8 gene. Arrows indicate the position of the c.176T>C, which resulted in substitution of Methionine (Met, M) to Threonine (Thr, T) at codon 59 (p.M59T). ( B ) Degree of conservation of the Met 59 residue (shaded) across different species. ( C ) Visual comparison of predicted models. ERCC8 WT is shown in pink color ribbons, while ERCC8 M59T is shown in sky blue color. The regions with conformational changes are labelled in black. Position of M59 is shown green in WT, while red in mutant structure.

Article Snippet: Overexpression vectors containing WT ERCC8 transcript variant 1 cDNA were purchased (Origene, Rockville, MD, USA; Cat# RC214392) and site-directed mutagenesis was performed (Agilent QuikChange II XL; Cat# 200521; Agilent Technologies, Santa Clara, CA, USA) to generate mutant ERCC8 expression vectors using following primers: forward 5′-TCTGAACCACCTGATAACGTGTATCTCCCTTCAACAG-3′ and reverse 5′-CTGTTGAAGGGAGATACACGTTATCAGGTGGTTCAGA-3′.

Techniques: Variant Assay, Sequencing, Mutagenesis, Residue, Comparison

Journal: Cells

Article Title: A Novel Missense Mutation in ERCC8 Co-Segregates with Cerebellar Ataxia in a Consanguineous Pakistani Family

doi: 10.3390/cells11193090

Figure Lengend Snippet: ERCC8 M59T mutation decreases protein stability. ( A ) Chromatograms of DNA sequence of plasmids to overexpress full-length ERCC8 WT (transcript variant 1) or full-length ERCC8 M59T . The highlighted nucleotides show the WT sequence (top) and the successfully mutated plasmid (bottom). ( B ) Schematic of ERCC8 protein stability assay. ( C , D ) Representative western blots ( C ) and quantification ( D ) of WT and mutant ERCC8 degradation over time ( n = 3 biological replicates). * p < 0.05, t -test.

Article Snippet: Overexpression vectors containing WT ERCC8 transcript variant 1 cDNA were purchased (Origene, Rockville, MD, USA; Cat# RC214392) and site-directed mutagenesis was performed (Agilent QuikChange II XL; Cat# 200521; Agilent Technologies, Santa Clara, CA, USA) to generate mutant ERCC8 expression vectors using following primers: forward 5′-TCTGAACCACCTGATAACGTGTATCTCCCTTCAACAG-3′ and reverse 5′-CTGTTGAAGGGAGATACACGTTATCAGGTGGTTCAGA-3′.

Techniques: Mutagenesis, Sequencing, Variant Assay, Plasmid Preparation, Stability Assay, Western Blot