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 Mean RPKM values for various examples of IRE1α RNase targets from the RNAseq and GROseq datasets in WT and IRE1α KO MDA-MB-231 cells before and after ER-stress induction by Tg (100 nM). Note that the GROseq data points in some cases are hidden behind the RNAseq data points. n = 3 biologically independent experiments. Data are presented as mean values ± SEM. An Unpaired t -test was used to calculate p -values. b Kinetic RT-qPCRs analysis of CD59 and TNFAIP8L1 transcripts in IRE1α WT and KO MDA-MB-231 cells, before and after ER-stress induction by Tg (100 nM) for 2 and 8 h. n = 3 biologically independent experiments. Data are presented as mean values ± SEM. An Unpaired t -test was used to calculate p -values. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: We prepared T7 RNA transcripts from cDNA templates chosen based upon functional relevance coupled with optimal length for the ribonucleolytic reaction (~0.5–2 kb). cDNA constructs encoding XBP1 (#HG10751-UT), DGAT2 (#HG14114-G), CD59 (#HG12474-UT), TGOLN2 (#HG17252-UT), SIX2 (#HG21116-UT), CFAP45 (#HG22377-UT), MFAP2 (#HG16644-UT), PIGQ (#HG22757-UT), BMP4 (#HG10609-UT), BCAM (#HG10238-UT), SNN (#HG23279-U), GBA (#HG12038-UT), WT1 (#HG12282-UT), CCDC69 (#HG27177-U), AIM2(#HG11654-UT) were from Sino Biological, and BLOC1S1 (#RC224412), TNFAIP8L1 (#RC203912) from Origene. cDNA was amplified using T7 forward primers, and subsequently in vitro transcribed using HiScribe™ T7 Quick High Yield RNA Synthesis Kit from NEB (#E2050S).

Techniques:

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: We prepared T7 RNA transcripts from cDNA templates chosen based upon functional relevance coupled with optimal length for the ribonucleolytic reaction (~0.5–2 kb). cDNA constructs encoding XBP1 (#HG10751-UT), DGAT2 (#HG14114-G), CD59 (#HG12474-UT), TGOLN2 (#HG17252-UT), SIX2 (#HG21116-UT), CFAP45 (#HG22377-UT), MFAP2 (#HG16644-UT), PIGQ (#HG22757-UT), BMP4 (#HG10609-UT), BCAM (#HG10238-UT), SNN (#HG23279-U), GBA (#HG12038-UT), WT1 (#HG12282-UT), CCDC69 (#HG27177-U), AIM2(#HG11654-UT) were from Sino Biological, and BLOC1S1 (#RC224412), TNFAIP8L1 (#RC203912) from Origene. cDNA was amplified using T7 forward primers, and subsequently in vitro transcribed using HiScribe™ T7 Quick High Yield RNA Synthesis Kit from NEB (#E2050S).

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

Journal: bioRxiv

Article Title: The sleep quality- and myopia-linked PDE11A-Y727C variant impacts neural physiology by reducing catalytic activity and altering subcellular compartmentalization of the enzyme

doi: 10.1101/2023.11.16.567422

Figure Lengend Snippet: The Y727C and M878V variants dramatically alter PDE11A4 subcellular localization in HT22 cells. A) Immunocytochemistry of untagged human PDE11A4 (hPDE11A4) or B) direct visualization of a GFP-tagged mouse PDE11A4 (mPDE11A4) shows that the Y727C and M878V variants disperse the enzyme. Quantification shows that C) Y727C and D) M878V reduce the occurrence of small mPDE11A4 puncta to the same extent as a previously reported phosphomimic mutation of serine 162 (S162D; ; each variant, n=18 biological replicates/group over 3 experiments). E) Y727C also increases the presence of larger mPDE11A4 punctate accumulations, while F) S162D and M878V do not. G-H) When the Y727C variant is co-expressed with PDE11A4-WT, as would occur in a patient, the phenotype of the Y727C variant completely dominates that of the WT construct, whereas WT+M878V results in the mathematically-predicted 50% effect size of M878V alone. I) Y727C decreases PDE11A4-pS117/pS124 (n=5/group) but J) M878V does not (n=4/group). K) Images of representative PDE11A4-pS117/pS124 (pp-11A4) and total PDE11A4 (t-11A4) Western blots. L) Neither a phosphomimic S117D/S124D nor a phosphoresistant S162A changed the ability of Y727C to reduce the presence of small PDE11A4 puncta nor M) increase the presence of large PDE11A4 accumulations. N) Similarly, S162A does not alter the dispersal effects of M878V; however, M878V is not able to fully block the pro-accumulating effect of S117D/S124D. O) There is no significant interaction between M878V and the phosphomutants on large PDE11A4 puncta. P) The phosphoresistant Y727A and the phosphomimic Y727D mutants elicited the same effect as the Y727C variant on P) small and Q) large PDE11A4 puncta. R) Relative to mCherry alone (i.e., negative control), overexpression of TGN38 increases the presence of small PDE11A4-WT, PDE11A4-Y727C, and PDE11A4-M878V puncta and S) doubles the effect of Y727C on the accumulation of large PDE11A4 puncta. Histogram stretch, brightness, and contrast adjusted for graphical clarity. Post hoc *vs. WT, P= 0.0448-

Article Snippet: Oligonucleotide primers were generated by Integrated DNA Technologies (Coralville, IA) and mutated DNA sequences were verified by Functional Biosciences (Madison, WI). mCherry in pcDNA3.1 was also generated by Genescript and the TGN38 plasmid was purchased from Origene.

Techniques: Immunocytochemistry, Mutagenesis, Variant Assay, Construct, Western Blot, Blocking Assay, Negative Control, Over Expression

Journal: iScience

Article Title: Compartmentalization of casein kinase 1 γ CSNK1G controls the intracellular trafficking of ceramide

doi: 10.1016/j.isci.2022.104624

Figure Lengend Snippet: CK1G3 localizes to cytoplasmic punctate compartments Intracellular localization of HA-CK1G3. CK1G3 KO cells expressing HA-CK1G3 were immunostained with HA antibody and antibodies against various organelle markers: LBPA (late endosomes), EEA1 (early endosomes), Lamp2 (lysosomes), Hrs (exosomes), VAP (ER), TGN46 (TGN), Rab11a (recycling endosomes and Rab11a-positive secretory vesicles), catalase (peroxisomes), TOMM20 (mitochondria), caveolin (caveolae), and lipid dye II (lipid droplet). For the visualization of lipid droplets, cells were pre-incubated in a medium containing 100 μM oleic acid for 24 h before fixation. The scale bars indicated in microscopy images represent 10 μm.

Article Snippet: Anti-HA antibody (#3F10) was purchased from Roche Diagnostics; anti-CERT (#ab72536) from Abcam; chicken antibody against VAP ( ) anti-TGOLN2/TGN46 (#A304-434A) from Bethyl Laboratories; anti-TOMM20 (#WH0009804M1) from Sigma-Aldrich; anti-Lamp2 (#sc-18822) from Santa Cruz; anti-LBPA (#z-PLBPA) from Echelon Bioscience; anti-EEA1 (#610457) from BD Transduction Laboratories; anti-catalase (#D4P7B) and anti-Rab11a (#2413) from Cell Signaling Technology; anti-Hrs (#10390-1-AP) from Proteintech; anti-GFP (#04404-84) from Nacalai Tesque, and anti-GAPDH (016-25523) from Fujifilm Wako Pure Chemical Corporation.

Techniques: Expressing, Incubation, Microscopy

Journal: iScience

Article Title: Compartmentalization of casein kinase 1 γ CSNK1G controls the intracellular trafficking of ceramide

doi: 10.1016/j.isci.2022.104624

Figure Lengend Snippet: Compartmentalization-dependent functional control of CK1Gs After newly synthesized as a cytosolic protein, CK1G presumably binds to the Golgi-membrane via the electrostatic interaction between its C -terminal basic amino acids stretch and the Golgi localizing PtdIns(4)P (see also the text) and subsequently palmitoylated by the Golgi-residing palmitoyl acyltransferase (PAT). Palmitoylated CK1G is largely distributed to post-Golgi compartments and only partly to the Golgi apparatus, where SM synthase 1 (SMS1) is localized. CERT is associated with the ER via VAP-binding and with the Golgi via PtdIns(4)P-binding. It remains elusive whether palmitoylated CK1Gs are recycled among the distal- and post-Golgi compartments or eventually directed to lysosomes (for degradation) or exosomes (for secretion). The Golgi-distributed CK1G is spatially limited to interact only with CERT recruited to the ER-Golgi contact zone. In contrast, the C -terminus deleted CK1Gs are distributed throughout the cytosol and can phosphorylate CERT anywhere in the cells once the priming phosphorylation of CERT S132 by PKD occurs.

Article Snippet: Anti-HA antibody (#3F10) was purchased from Roche Diagnostics; anti-CERT (#ab72536) from Abcam; chicken antibody against VAP ( ) anti-TGOLN2/TGN46 (#A304-434A) from Bethyl Laboratories; anti-TOMM20 (#WH0009804M1) from Sigma-Aldrich; anti-Lamp2 (#sc-18822) from Santa Cruz; anti-LBPA (#z-PLBPA) from Echelon Bioscience; anti-EEA1 (#610457) from BD Transduction Laboratories; anti-catalase (#D4P7B) and anti-Rab11a (#2413) from Cell Signaling Technology; anti-Hrs (#10390-1-AP) from Proteintech; anti-GFP (#04404-84) from Nacalai Tesque, and anti-GAPDH (016-25523) from Fujifilm Wako Pure Chemical Corporation.

Techniques: Functional Assay, Control, Synthesized, Membrane, Binding Assay, Phospho-proteomics

Journal: iScience

Article Title: Compartmentalization of casein kinase 1 γ CSNK1G controls the intracellular trafficking of ceramide

doi: 10.1016/j.isci.2022.104624

Figure Lengend Snippet:

Article Snippet: Anti-HA antibody (#3F10) was purchased from Roche Diagnostics; anti-CERT (#ab72536) from Abcam; chicken antibody against VAP ( ) anti-TGOLN2/TGN46 (#A304-434A) from Bethyl Laboratories; anti-TOMM20 (#WH0009804M1) from Sigma-Aldrich; anti-Lamp2 (#sc-18822) from Santa Cruz; anti-LBPA (#z-PLBPA) from Echelon Bioscience; anti-EEA1 (#610457) from BD Transduction Laboratories; anti-catalase (#D4P7B) and anti-Rab11a (#2413) from Cell Signaling Technology; anti-Hrs (#10390-1-AP) from Proteintech; anti-GFP (#04404-84) from Nacalai Tesque, and anti-GAPDH (016-25523) from Fujifilm Wako Pure Chemical Corporation.

Techniques: Transduction, Recombinant, Protease Inhibitor, Software

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