Journal: JCI Insight

Article Title: A metabolic redox relay supports ER proinsulin export in pancreatic islet β cells

doi: 10.1172/jci.insight.178725

Figure Lengend Snippet: Male and female 12- to 16-week-old mouse islets ( A – C ) or INS-1 832/3 cells ( D and E ) were treated with vehicle (veh) or mannoheptulose (MnH; 2 mM or 1 mM, respectively) for 4 hours as indicated. NADPH/NADP + ( A , n = 4) and GSSG/GSH ( B , n = 4) were measured by sequential incubation in 2 mM Glc followed by 20 mM Glc for 12 minutes each via ratiometric imaging of iNAP or Grx1-roGFP2 (AdRIP), respectively. Responses were normalized to 2 mM Glc. ( C ) ER redox was measured ( n = 5) via ratiometric imaging of ERroGFP (AdRIP). ( D and E ) INS-1 832/3 cells stably expressing proCpepSNAP cells were treated with veh or MnH or cotreated with MnH plus DTT (0.5 mM; MnH + DTT) for 4 hours as indicated. Cells were pulse-labeled with SNAP-505 (green), chased for 10 minutes, immunostained for TGN38 (magenta) and BiP (red), and counterstained with DAPI (blue). ( D ) The ratio of proCpepSNAP fluorescence coincident with the Golgi (TGN38) versus ER (BiP) was quantified ( n = 4). ( E ) Representative images are shown. ( A – D ) Data represent the mean ± SEM. * P < 0.05 by 2-tailed Student’s t test ( A – C ) or 1-way ANOVA with Tukey’s posttest analysis ( D ). Scale bar = 5 μm.

Article Snippet: For immunostaining, cells were incubated overnight with antibodies raised against BiP (rabbit; gift of Christopher Nicchitta, Duke University, Durham, North Carolina, USA; 1:200), GRASP55 (rabbit, Proteintech 10598-AP, 1:500), GM130 (mouse, BD Transduction 610-823, 1:200), TGN38 (mouse, Novus Biologicals, Bio-Techne, NB300-575, 1:200), or proinsulin (mouse, MyBioSource MBS660187, 1:200) as indicated.

Techniques: Incubation, Imaging, Stable Transfection, Expressing, Labeling, Fluorescence

Journal: JCI Insight

Article Title: A metabolic redox relay supports ER proinsulin export in pancreatic islet β cells

doi: 10.1172/jci.insight.178725

Figure Lengend Snippet: ( A and B ) Male and female 12- to 16-week-old mouse islets or INS-1 832/3 cells were treated with vehicle (veh), 2-AAPA (25 μM), or auranofin (AFN; 10 μM) for 4 hours before imaging. ( A ) NADPH/NADP + were measured in islets ( n = 4) by sequential incubation in 2 mM Glc followed by 20 mM Glc for 12 minutes each via ratiometric imaging of iNAP (AdRIP). Responses were normalized to 2 mM Glc. ( B ) INS-1 832/3 cells stably expressing proCpepSNAP were pulse-labeled with SNAP-505, chased for 10 minutes, immunostained for BiP and TGN38, and counterstained with DAPI. The ratio of proCpepSNAP fluorescence coincident with the Golgi (TGN38) versus the ER (BiP) was quantified ( n = 3). ( C – G ) Male and female 12- to 16-week-old mouse islets were treated with Ad-shSAFE or Ad-sh Txnrd1 as indicated and analyzed 96 hours after infection. ( C ) Txnrd1 mRNA expression was quantified by RT-qPCR ( n = 3–4). ( D , E , and G ) Male and female 12- to 16-week-old mouse islets expressing proCpepSNAP (AdRIP) were pulse-labeled with SNAP-505 (green) and chased for 10 minutes. Cells were fixed, immunostained for GM130 (magenta), and counterstained for DAPI (blue). Representative images are shown ( D ) and the ratio of proCpepSNAP coincident with the Golgi (GM130) versus non-Golgi region in mCherry + cells ( n = 5) was quantified ( E ) and total fluorescence intensity calculated ( G ). ( F ) ER redox was measured in mCherry + islet cells (Ad-shRNA; n = 4) via ratiometric imaging of ERroGFP (AdRIP). ( A – C and E – G ) Data represent the mean ± SEM. * P < 0.05 by 1-way ANOVA with Dunnett’s posttest analysis ( A ), 1-way ANOVA with Tukey’s posttest analysis ( B ), or 2-tailed Student’s t test ( C and E – G ). Scale bar = 5 μm.

Article Snippet: For immunostaining, cells were incubated overnight with antibodies raised against BiP (rabbit; gift of Christopher Nicchitta, Duke University, Durham, North Carolina, USA; 1:200), GRASP55 (rabbit, Proteintech 10598-AP, 1:500), GM130 (mouse, BD Transduction 610-823, 1:200), TGN38 (mouse, Novus Biologicals, Bio-Techne, NB300-575, 1:200), or proinsulin (mouse, MyBioSource MBS660187, 1:200) as indicated.

Techniques: Imaging, Incubation, Stable Transfection, Expressing, Labeling, Fluorescence, Infection, Quantitative RT-PCR, shRNA

Journal: Nature

Article Title: Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes

doi: 10.1038/s41586-023-06857-0

Figure Lengend Snippet: a , b , Immunoblots depicting expression levels of ApoB48 in SI IECs ( a ) and ApoB48 and ApoB100 on TRLs isolated from plasma by ultracentrifugation ( b ) from Dars2 fl/fl and Dars2 tamIEC-KO mice 5 and 7 days after the last tamoxifen injection ( a , n = 4 mice per genotype, per indicated time point; b , n = 5 mice per genotype at 7 days after tamoxifen, n = 4 mice per genotype at 5 days after tamoxifen). γ-tubulin was used as the loading control ( a ). AT, after tamoxifen. c , Representative TEM micrographs from proximal SI sections of Dars2 fl/fl mice ( n = 4) and Dars2 tamIEC-KO mice ( n = 4) 7 days after the last tamoxifen injection. Note the lack of CM-containing Golgi complexes and the appearance of aberrant numbers of LDs and damaged mitochondria in DARS2-deficient enterocytes. Asterisks indicate CMs secreted in the basolateral intercellular space. Arrows point at lipid particles within the ER lumen in rough and smooth ER neighbouring areas. Arrowheads point at LD lateral fusion. N, nucleus. d , Top, representative fluorescence microscopy images from the proximal SI of 8–12-week-old Dars2 fl/fl mice ( n = 6) and Dars2 tamIEC-KO mice ( n = 6) immunostained with antibodies against TGN38, E-cadherin and PLIN2. Nuclei stained with DAPI. Arrowheads point at LDs. Bottom, quantification of the TGN38-positive puncta size and the number of puncta per nucleus from confocal images of proximal SI sections of Dars2 fl/fl mice ( n = 4) and Dars2 tamIEC-KO mice ( n = 5) 5 days after the last tamoxifen injection. In d , dots represent individual mice, bar graphs show the mean ± s.e.m. and P values were calculated using unpaired two-sided Student’s t -test with no assumption of equal variance. In a , b , each lane represents one mouse from two independent experiments. Scale bars, 1 μm ( c ) or 50 μm ( d ). For gel source data, see Supplementary Fig. .

Article Snippet: Immunofluorescence staining was performed on IEC-6 cells cultured on coverslips and fixed in 4% paraformaldehyde for 15 min. Reactive aldehydes were quenched with 50 mM NH 4 Cl for 10 min and the cells were permeabilized with 0.1% Triton-X-100 in PBS for 5 min. After 20 min in blocking solution (0.2% fish-skin gelatin diluted in PBS), IEC-6 cells were incubated with primary antibodies against TGN38 (bio-techne, AF8059-SP, 1:200) and MTCO1/COX1 (Molecular Probes, 459600, 1D6E1A8, 1:100) for 30 min at room temperature, followed by incubation with anti-sheep IgG NorthernLights NL557 (bio-techne, NL010, 1:300) or anti-mouse Alexa 488 (Molecular Probes, A1101, 1:300) fluorescence-conjugated secondary antibodies for 30 min at room temperature.

Techniques: Western Blot, Expressing, Isolation, Injection, Fluorescence, Microscopy, Staining

Journal: Nature

Article Title: Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes

doi: 10.1038/s41586-023-06857-0

Figure Lengend Snippet: a , Representative fluorescence microscopy images from the proximal SI of 8-12-week-old Dars2 fl/fl ( n = 6) and DARS2 tamIEC-KO ( n = 6) mice sacrificed 3, 5 and 8 days upon the last tamoxifen injection and immunostained with antibodies against TGN38 (red) and E-cadherin (green). Insets shows only TGN38 staining in white. b , Representative fluorescence microscopy images from the proximal and distal SI of 8-12-week-old Dars2 fl/fl ( n = 6) and DARS2 tamIEC-KO ( n = 6) mice fed with NCD sacrificed 3 and 8 days upon the last tamoxifen injection and immunostained with antibodies against TGN38 (red), E-cadherin (green) and PLIN2 (yellow). c , Representative fluorescence microscopy images from the proximal SI of 8-12-week-old Dars2 fl/fl ( n = 6) and DARS2 tamIEC-KO ( n = 6) mice under NCD and FFD sacrificed 7 days upon the last tamoxifen injection and immunostained with antibodies against TGN38 (red), E-cadherin (green) and PLIN2 (yellow). Nuclei stained with DAPI (blue). Scale bars, 50 μm. Confocal images shown are representative of the number of mice analysed as indicated in Supplementary Table .

Article Snippet: Immunofluorescence staining was performed on IEC-6 cells cultured on coverslips and fixed in 4% paraformaldehyde for 15 min. Reactive aldehydes were quenched with 50 mM NH 4 Cl for 10 min and the cells were permeabilized with 0.1% Triton-X-100 in PBS for 5 min. After 20 min in blocking solution (0.2% fish-skin gelatin diluted in PBS), IEC-6 cells were incubated with primary antibodies against TGN38 (bio-techne, AF8059-SP, 1:200) and MTCO1/COX1 (Molecular Probes, 459600, 1D6E1A8, 1:100) for 30 min at room temperature, followed by incubation with anti-sheep IgG NorthernLights NL557 (bio-techne, NL010, 1:300) or anti-mouse Alexa 488 (Molecular Probes, A1101, 1:300) fluorescence-conjugated secondary antibodies for 30 min at room temperature.

Techniques: Fluorescence, Microscopy, Injection, Staining

Journal: Nature

Article Title: Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes

doi: 10.1038/s41586-023-06857-0

Figure Lengend Snippet: a , Representative fluorescence microscopy images depicting IEC-6 cells treated for 48 h with actinonin (100 μM), Atpenin A5 (AA5, 1 μ M) or 1% dimethyl sulfoxide; DMSO (control). Short treatment (6 h) with Brefeldin A (BFA, 5 μg/ml) was used as a positive control for Golgi dispersal. Scale bars, 50 μm b , Representative fluorescence microscopy images of IEC-6 cells grown under the same conditions as described in a were incubated with oleic acid (OA, 600 μM) for the last 24 h prior imaging. In this case, BFA was applied in the last 6 h of OA treatment to avoid cytotoxicity. Anti-TGN38 (red) antibody was used to visualize Golgi, Anti-COX1(green) to stain mitochondrial networks (a) , BODIPY (green) to stain lipid droplets (b) , and DAPI (blue) for nuclei (top). TGN38 staining is additionally depicted in white (bottom). Scale bars, 50 μm. Quantification of the observed Golgi morphology of the IEC-6 cells based on five distinct categories, as illustrated at the right ( n = 100–300 inspected IEC-6 cells from three independent biological experiments). c , Representative confocal images and graphs depicting quantification of GFP signal in C. elegans expressing α-mannosidase II fused to GFP under the control of the gut-specific vha-6 promoter grown either on a control empty vector (EV) or RNAi against dars-2 at the first (D1) and fourth day of adulthood (D4) (EV, n = 19 ( D1 ), n = 13 ( D4 ), dars-2 , n = 20 ( D1 ), n = 19 ( D4 )). Scale bars, 1 μm. d , Representative confocal images of GFP signal in C. elegans expressing SPCS-1 fused to GFP under the control of the gut-specific vha-6 promoter grown either on a control empty vector (EV) or RNAi against dars-2 at the first (D1) and fourth day of adulthood (D4) EV, n = 19 ( D1 ), n = 19 ( D4 ), dars-2 , n = 20 ( D1 ), n = 19 ( D4 )). Scale bars, 1 μm. e , Immunofluorescence micrographs of C. elegans carrying vit-2::GFP reporter on a control empty vector (EV) ( n = 10) and RNAi against sar-1 , sec-13 , fum-1 and dars-2 at D1 ( n = 10). Insets show magnification of a selected area of the worm. Scale bars, 100 μm. In bar graphs data are represented as mean ± s.e.m. and P values were calculated by two-sided Chi-squared test ( a , b ) and two-sided Student’s t -test with assumption of equal variance ( c ).

Article Snippet: Immunofluorescence staining was performed on IEC-6 cells cultured on coverslips and fixed in 4% paraformaldehyde for 15 min. Reactive aldehydes were quenched with 50 mM NH 4 Cl for 10 min and the cells were permeabilized with 0.1% Triton-X-100 in PBS for 5 min. After 20 min in blocking solution (0.2% fish-skin gelatin diluted in PBS), IEC-6 cells were incubated with primary antibodies against TGN38 (bio-techne, AF8059-SP, 1:200) and MTCO1/COX1 (Molecular Probes, 459600, 1D6E1A8, 1:100) for 30 min at room temperature, followed by incubation with anti-sheep IgG NorthernLights NL557 (bio-techne, NL010, 1:300) or anti-mouse Alexa 488 (Molecular Probes, A1101, 1:300) fluorescence-conjugated secondary antibodies for 30 min at room temperature.

Techniques: Fluorescence, Microscopy, Positive Control, Incubation, Imaging, Staining, Expressing, Plasmid Preparation, Immunofluorescence

Journal: Function

Article Title: ER Redox Homeostasis Regulates Proinsulin Trafficking and Insulin Granule Formation in the Pancreatic Islet β-Cell

doi: 10.1093/function/zqac051

Figure Lengend Snippet: Delayed ER export of proinsulin in animal and cell culture models of hyperglycemia. (A–C) INS1 832/3 cells stably expressing proCpepSNAP were cultured for up to 72 h in control media supplemented with BSA or media containing oleate/palmitate (2:1, 1 m m ) and elevated glucose (20 m m ; OPG) as indicated. Cells were pulse-labeled with SNAP-505 (green), chased for 15 min, immunostained for GRP94 (red), TGN38 (magenta), and counterstained with DAPI (blue). Representative images (A) are shown (scale bar = 5 μm) and the ratio of proCpepSNAP fluorescence coincident with the Golgi compared to ER quantified (B) ( n = 6–8 independent experiments; 48–64 cells per condition). (C) Total SNAP fluorescence per cell was quantified. (D and E) Isolated mouse islets (C57BLKS/J db/+ vs. db/db) were treated with AdRIP-proCpepSNAP. 48 h post-infection, islets were pulse-labeled with SNAP-505 (green), chased for 10 min, immunostained for GM130 (magenta), and counterstained with DAPI (blue). Representative images (E) are shown (scale bar = 3 μ m ) and the ratio of proCpepSNAP fluorescence coincident with the Golgi compared to ER quantified (D) ( n = 4 mice per group; 6–36 cells/mouse). (B and D) Data represent the mean ± SD * P < 0.05, *** P < 0.0005, or not significant (ns) by Student’s t -test.

Article Snippet: For immunostaining, cells were incubated overnight with antibodies raised against GM130 (mouse, BD Transduction 610 823, 1:200), GRP94 (rabbit, kind gift of Dr. Christopher Nicchita, Duke University, 1:500), or TGN38 (mouse, Novus Biologicals NB300-575, 1:200) as indicated.

Techniques: Cell Culture, Stable Transfection, Expressing, Control, Labeling, Fluorescence, Isolation, Infection

Journal: Function

Article Title: ER Redox Homeostasis Regulates Proinsulin Trafficking and Insulin Granule Formation in the Pancreatic Islet β-Cell

doi: 10.1093/function/zqac051

Figure Lengend Snippet: Proinsulin ER export delay can be rescued by chemical reducing agent. INS1 832/3 cells stably expressing proCpepSNAP were cultured in control media supplemented with BSA or media containing oleate: palmitate (2:1 and 1 m m ) and elevated glucose (20 m m ; OPG) as indicated. (A-C) Prior to SNAP labeling, cells were treated with vehicle (control) or DTT (0.5 m m ) for 4 h. Cells were then pulse-labeled with SNAP-505 (green), fixed, and immunostained for GRP94 (red), TGN38 (magenta), and counterstained with DAPI (blue). Representative images (A) are shown (scale bar = 5 μ m ) and Mander’s correlation coefficient (B) was used to determine the colocalization of labeled proCpepSNAP (SNAP) with TGN38 ( n = 3–4 independent experiments; 71–91 cells/condition). Total SNAP fluorescence was quantified (C). (D) Cells expressing ERroGFP (AdRIP) were cultured overnight as indicated with vehicle (control) or ebselen (Eb, 10 μ m ) and imaged at 470/535 nm and 395/510 nm. Normalized ratiometric intensities were quantified to determine ER oxidation ( n = 4 independent experiments). (E) Cells were cultured overnight as indicated with vehicle (-) or ebselen (Eb, 10 μM). GSIS was measured by static incubation in media containing 2.5 m m Glc followed by 12 m m Glc for 1 h each. (B–E) Data represent the mean ± SD * P < 0.05 by two-way ANOVA with Sidak post-test analysis (B, D, and E), or ns by two-way ANOVA (C).

Article Snippet: For immunostaining, cells were incubated overnight with antibodies raised against GM130 (mouse, BD Transduction 610 823, 1:200), GRP94 (rabbit, kind gift of Dr. Christopher Nicchita, Duke University, 1:500), or TGN38 (mouse, Novus Biologicals NB300-575, 1:200) as indicated.

Techniques: Stable Transfection, Expressing, Cell Culture, Control, Labeling, Fluorescence, Incubation

Journal: Nature Communications

Article Title: Decoding non-canonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α

doi: 10.1038/s41467-021-27597-7

Figure Lengend Snippet: a Western blot analysis of endogenous and ectopic IRE1α variant expression in MDA-MB-231 cells harboring Dox-inducible IRE1α shRNA stably transfected with transgenic WT or R887A mutant versions of IRE1α-GFP. b Immunoblot analysis of MDA-MB-231 cells after treatment with Tg (100 nM, 4 h) followed by DSS crosslinking. Left panel shows parental shIRE1α cl.12 cell line. Right panel shows IRE1α WT and R887A rescues of Doxycycline-treated cl.12 cells with endogenous IRE1α knockdown. c RT-qPCR analysis of IRE1α RNase targets CD59, TGOLN2 (RIDD), and TNFAIP8L1, SNN, and SIX2 (RIDDLE). Ct values for XBP1 in sample shIRE1 cl.1 prior to Tg treatment were >34, precluding ratio calculations and were therefore not plotted. n = 3 biologically independent experiments. Data are presented as mean values ± SEM. A 2-way ANOVA test was used to calculate p-values for CD59 and TGOLN2, and an unpaired t -test for the remaining targets. d Analysis of cell viability by Cell-Titer Glo after Dox treatment for 7 days on Ultra-Low Attachment (ULA) plates. n = 2 biologically independent experiments. Data are presented as mean values ± SEM. e Model depicting IRE1α’s principal modes of endoribonuclease function and their underlying phospho-oligomeric states during ER stress. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: Antibodies (Abs) for IRE1α (#3294), TGOLN2 (#95649), AIM2 (#12948), Actin (#5125), and GAPDH (#8884) from Cell Signaling Technology.

Techniques: Western Blot, Variant Assay, Expressing, shRNA, Stable Transfection, Transfection, Transgenic Assay, Mutagenesis, Knockdown, Quantitative RT-PCR