Journal: Shock

Article Title: Modeling Acute ER Stress in Vivo and in Vitro

doi: 10.1097/shk.0000000000000759

Figure Lengend Snippet: FIG. 3. No difference in ER stress induction in the epididymal fat of TG or TUN mice. Male Balb/c mice were injected with either control buffer (Con) or thapsigargin (TG) tunicamycin (Tun). After 24 h, the epididymal fat pads were dissected and (A) equal amounts of protein were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing ATF-6, eIF2a, and IRE1a. B, Densitometry quantification of ER stress proteins expressed over loading control a/b tubulin. Data shown are in mean SEM, *P <0.05 versus sham, # P <0.05 versus TG (n ¼ 3–4). ATF-6 indicates activating transcription factor 6; eIF2a, eukaryotic translation initiator factor 2a; IRE1a, inositol-requiring enzyme.

Article Snippet: Proteins were resolved by SDS-PAGE followed by Western blotting using antibodies recognizing GRP78 (BiP), inositol-requiring enzyme (IRE1a), eukaryotic initiation factor (eIF2a), CCAAT-enhancer-binding protein (CHOP), alpha/ beta tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), isocitrate dehydrogenases (IDH1), and activating transcription factor 6 (ATF-6) (Novus Biologicals, Littleton, Colo).

Techniques: Injection, Control, SDS Page, Western Blot

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A-D ) Depiction of zebrafish eif2b subunits exon structure and the location and nucleotide change for each mutant. ( A ) eif2b1 harbors a T/A transversion resulting in an early stop in exon 8. ( B ) eif2b2 has a G/A transition in exon 5, mutating an essential splice site. ( C ) eif2b4 has a G/A transition in exon 12 mutating an essential splice site. ( D ) eif2b5 exon one was targeted for mutagenesis using a gRNA (red). Six distinct alleles were recovered (described in text). ( E ) Chromatograms of cDNA confirm presence of predicted mutations for eif2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 . ( F ) eif2b5 zc103/zc103 mutants survive until adulthood in Mendelian ratios, but show grow defects compared to their heterozygous and wild-type siblings. ( G ) Adult eif2b5 zc103/zc103 lengths are significantly shorter compared to their wild-type and heterozygous siblings. ( H ) Bright-field (BF) images of 6 dpf eif2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 larva. eif2b2 sa17223/sa17223 and eif2b5 zc102/zc102 have no swim bladder (arrowhead) and a small head. ( I ) Kaplan-Meyer survival curves from an eif2b2 sa17223/+ heterozygous in-cross shows 1% (n = 3) homozygote survival at 10 dpf (total n = 302); however no homozygotes live past 2 weeks of age. ( J ) Kaplan-Meyer survival curves from an eif2b5 zc102/+ heterozygous in-cross shows that all homozygotes were dead by 10 dpf (total n = 62). ( K ) Kaplan-Meyer survival curves from an eif2b5 zc103/+ heterozygous in-cross show no mortality of homozygotes. ( L ) Motor swimming analysis shows impaired swimming behavior in mutants. Distance moved, time spent moving, and velocity, for wild-type controls, and eif2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 mutants, at 5, 6, and 7 dpf. Mean shown with 95% confidence intervals. Figure 2—source data 1. Quantification of lengths. Figure 2—source data 2. Quantification of behavior results. Figure 2—source data 3. Quantification of behavior results of eif2b4 sa17367/sa17367 allele. Figure 2—source data 4. qRT-PCR for ISR transcripts for eif2b4 sa17367/sa17367 allele. Figure 2—source data 5. Survival quantification for eif2b4 sa17367/sa17367 allele. Figure 2—source data 6. Survival quantification for eif2b5 zc103/zc103 allele.

Article Snippet: We tried western blotting with several commercial antibodies against Eif2b2 and Eif2b5 (Eif2B2 Abcam 133848; Eif2B5 Abcam ab91563, GeneTex 30808, Santa Cruz 514056, ProSci 56–847, Bethyl A302-557A) but none gave a specific band.

Techniques: Mutagenesis, Quantitative RT-PCR

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: Confocal images, z-stack maximal projections. ( A–I ), dorsal views of the brain, rostral to the top, scale bar 50 μm; ( K–N ), lateral views of spinal cord, dorsal to the top, scale bar 50 um. ( P–S ), dorsal views of brain, rostral to top, scale bar 50 μm. *p<0.05. ( A–D ) TUNEL and DAPI staining shows increased apoptosis in homozygous mutant alleles compared to controls (wild-type and heterozygous siblings) in ei f2b5 zc103/zc103 eif2b5 zc102/zc102 and eif2b2 sa17223/sa17223 mutants. ( E ) Quantification of mean TUNEL+ cell counts in ei f2b5 zc103/zc103 eif2b5 zc102/zc102 and eif2b2 sa17223/sa17223 mutants compared to sibling controls. ( F–I ) Phospho-histone 3 and DAPI staining shows decreased cell proliferation in 5 dpf eif2b2 sa17223/sa17223 mutants compared to controls, while ei f2b5 zc103/zc103 eif2b5 zc102/zc102 mutants show a change in proliferation pattern, specifically in the optic tectum. ( J ) Quantification of mean number pH3+ cells counts in ei f2b5 zc103/zc103 eif2b5 zc102/zc102 and eif2b2 sa17223/sa17223 mutants compared to sibling controls. ( K–N ) Olig2dsRed and DAPI staining shows no change in OPC counts in the spinal cords of 5 dpf ei f2b5 zc103/zc103 eif2b5 zc102/zc102 and eif2b2 sa17223/sa17223 mutants compared to controls. ( O ) Quantification of mean number Olig2dsRed+ counts in ei f2b5 zc103/zc103 eif2b5 zc102/zc102 or eif2b2 sa17223/sa17223 mutants compared to sibling controls. ( P–S ) Co-labeled Olig2dsRed+/TUNEL+ cell counts staining shows increase in Olig2dsRed+ cells undergoing apoptosis in brains of 5 dpf ei f2b5 zc103/zc103 , eif2b5 zc102/zc102 , or eif2b2 sa17223/sa17223 mutants compared to sibling controls. ( O ) Quantification of mean number of co-labeled Olig2dsRed+/TUNEL+ cell counts in ei f2b5 zc103/zc103 , eif2b5 zc102/zc102 , or eif2b2 sa17223/sa17223 mutants compared to sibling controls. Figure 3—source data 1. Quantification of TUNEL, pH3, olig2, and olig2/TUNEL results. Figure 3—source data 2. Quantification of TUNEL, pH3, olig2, and olig2/TUNEL results. Figure 3—source data 3. Quantification of TUNEL, pH3, olig2, and olig2/TUNEL results. Figure 3—source data 4. Quantification of TUNEL, pH3, olig2, and olig2/TUNEL results.

Article Snippet: We tried western blotting with several commercial antibodies against Eif2b2 and Eif2b5 (Eif2B2 Abcam 133848; Eif2B5 Abcam ab91563, GeneTex 30808, Santa Cruz 514056, ProSci 56–847, Bethyl A302-557A) but none gave a specific band.

Techniques: TUNEL Assay, Staining, Mutagenesis, Labeling

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: Confocal images of brain, z-stack, rostral to the top, double-labeling for TUNEL and olig1 (in situ probe), in WT, eif2b5 zc103/zc103 , eif2b5 zc102/zc102 or eif2b2 sa17223/sa172233 larvae. Region used for quantification shown by box. Inset in each panel shows example of double-labeled cell (TUNEL, olig1 ) for each genotype (except WT), single confocal slice image. Middle panels: no change in apoptosis of differentiated oligodendrocytes, co-labeled with myelin associated glycoprotein ( mag ) (in situ probe) and TUNEL. Confocal z-stack images of spinal cord, rostral to the left; quantified in lower right panel. Figure 4—source data 1. Quantification of olig1 , TUNEL and mag results.

Article Snippet: We tried western blotting with several commercial antibodies against Eif2b2 and Eif2b5 (Eif2B2 Abcam 133848; Eif2B5 Abcam ab91563, GeneTex 30808, Santa Cruz 514056, ProSci 56–847, Bethyl A302-557A) but none gave a specific band.

Techniques: Labeling, TUNEL Assay, In Situ

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A ) Confocal images of brain, z-stack, rostral to the top, in WT, eif2b5 zc103/zc103 , eif2b5 zc102/zc102 or eif2b2 sa17223/sa172233 larvae. Confocal images of brain, z-stack, rostral to the top. Top row, labeling for Zrf1, DAPI, and pH3. Middle row, labeling for HuC/D, DAPI, and pH3. Bottom row, labeling for apoeb , DAPI, and pH3. ( B ) Quantification of cell counts in ( A ). ( C ) Confocal images of brain, z-stack, rostral to the top, in WT, eif2b5 zc103/zc103 , eif2b5 zc102/zc102 or eif2b2 sa17223/sa172233 larvae. Confocal images of brain, z-stack, rostral to the top. Top row, labeling for Zrf1, DAPI, and TUNEL. Middle row, labeling for HuC/D, DAPI, and TUNEL. Bottom row, labeling for apoeb , DAPI, and TUNEL. ( D ) Quantification of cell counts in ( C ). Figure 5—source data 1. Quantification of Zrf1, HuC/D, and apoeb , double-labeling with pH3, results in tectum, midline, and eyes in eif2b5 zc103/zc103 , eif2b2 zc102/zc102 , and eif2b2 sa17223/sa172233 . Figure 5—source data 2. Quantification of Zrf1, HuC/D, and apoeb , double-labeling with TUNEL, results in tectum, midline, and eyes in eif2b5 zc103/zc103 , eif2b2 zc102/zc102 , and eif2b2 sa17223/sa172233 .

Article Snippet: We tried western blotting with several commercial antibodies against Eif2b2 and Eif2b5 (Eif2B2 Abcam 133848; Eif2B5 Abcam ab91563, GeneTex 30808, Santa Cruz 514056, ProSci 56–847, Bethyl A302-557A) but none gave a specific band.

Techniques: Labeling, TUNEL Assay

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A ) 11.1 kb zebrafish eif2b2 gene structure. ( B ) 9.5 kb human EIF2B2 gene structure. ( C ) Conservation of amino acid sequence between zebrafish eif2b2 and human EIF2B2. ( D ) Schematic of rescue construct containing Tol2, β-actin, human EIF2B2, and eGFP. ( E ) Genotype results (at adult age), of an eif2b2 sa17223/+ heterozygous incross, and an eif2b2 sa17223/+ heterozygous zebrafish crossed with two different transgenic alleles (#3 and #4): eif2b2 sa17223/+ ;Tg 3 ( β-actin:EIF2B2:2A:eGFP ) or eif2b2 sa17223/+ ;Tg 4 ( β-actin:EIF2B2:2A:eGFP ) heterozygous zebrafish. ( F ) Bright-field and immunofluorescent images of eif2b2 sa17223/sa17223 ;β-actin:EIF2B2:2A:eGFP mutant fish: showing a swim bladder and regular-sized head; or showing GFP expression in eif2b2 sa17223/sa17223 ;β-actin:EIF2B2:2A:eGFP mutant fish. ( G–J ) TUNEL and DAPI antibody staining of wild-type control; e if2b2 sa17223/sa17223 mutant; eif2b2 +/+ ;β-actin:EIF2B2:2A:eGFP wild-type control; and eif2b2 sa17223/sa17223 ;β-actin:EIF2B2:2A:eGFP mutant. ( K ) Quantification of TUNEL+ cells. Figure 7—source data 1. Quantification of TUNEL results. Figure 7—source data 2. Quantification of behavior results.

Article Snippet: We tried western blotting with several commercial antibodies against Eif2b2 and Eif2b5 (Eif2B2 Abcam 133848; Eif2B5 Abcam ab91563, GeneTex 30808, Santa Cruz 514056, ProSci 56–847, Bethyl A302-557A) but none gave a specific band.

Techniques: Sequencing, Construct, Transgenic Assay, Mutagenesis, Expressing, TUNEL Assay, Staining, Control

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A ) Schematic showing integrated stress response activated intron 12 retention of eif2b5 resulting in premature stop codon and truncated form of EIF2B5. ( B ) Fold change of eif2b5 intron 12 expression with qRT-PCR in ei f2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 mutants relative to controls. ( C ) qRT-PCR for intron 12 expression in a VWM patient and their control unaffected father. ( D ) Control-injected larvae, compared to those injected with truncated eif2b5 construct, shows impaired swimming behavior; distance moved, time spent moving, and velocity, at 5 dpf. ( E ) qRT-PCR for atf4 , bip , chopII , and perk ISR transcripts shows increased expression in ei f2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 mutants. ( F ) qRT-PCR for ISR transcripts ( atf 4 , bip , chop II , and perk ) shows increased expression following injection of truncated eif2b5 construct. Figure 8—source data 1. qRT-PCR for intron 12 expression changes ( eif2b5 , zebrafish). Figure 8—source data 2. qRT-PCR for intron 12 expression changes ( eif2b2 , zebrafish). Figure 8—source data 3. qRT-PCR for intron 12 expression changes (human). Figure 8—source data 4. Behavior data for truncated eif2b5 effects. Figure 8—source data 5. qRT-PCR for ISR transcript expression. Figure 8—source data 6. qRT-PCR for ISR transcript expression changes following injection with truncated eif2b5 .

Article Snippet: We tried western blotting with several commercial antibodies against Eif2b2 and Eif2b5 (Eif2B2 Abcam 133848; Eif2B5 Abcam ab91563, GeneTex 30808, Santa Cruz 514056, ProSci 56–847, Bethyl A302-557A) but none gave a specific band.

Techniques: Expressing, Quantitative RT-PCR, Control, Injection, Construct

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet:

Article Snippet: We tried western blotting with several commercial antibodies against Eif2b2 and Eif2b5 (Eif2B2 Abcam 133848; Eif2B5 Abcam ab91563, GeneTex 30808, Santa Cruz 514056, ProSci 56–847, Bethyl A302-557A) but none gave a specific band.

Techniques: Generated, Recombinant, Transfection, Construct, Labeling, Plasmid Preparation, SYBR Green Assay, Software, Staining

Journal: Nature Communications

Article Title: Reentrant DNA shells tune polyphosphate condensate size

doi: 10.1038/s41467-024-53469-x

Figure Lengend Snippet: Plasmids used in our study

Article Snippet: 30 kb , pLD1 translation factors , Addgene #117760 , 30,152 , BLR (F- ompT hsdSB(rB- mB-)gal dcm (DE3) Δ(srl-recA)306::Tn10 (TetR)).

Techniques: Plasmid Preparation, Lambda DNA Preparation

Journal: bioRxiv

Article Title: Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

doi: 10.1101/2021.09.29.462463

Figure Lengend Snippet: (A) EB1N-mApple-LOV2-labelled MT plus ends before and during blue light exposure in MG132-arrested metaphase spindles. Maximum intensity projections in alternating colors over 20 s at 3-s intervals and MT growth tracks over the same time window illustrate MT growth dynamics before and during blue light exposure. The comparison of the median MT growth rate per cell before and during blue light exposure is shown on the right. Grey lines connect data from the same cell. Statistical analysis by paired Student’s t-test. (B) Time-lapse sequence of a π-EB1 H1299 cell expressing mCherry-tagged α-tubulin. Note the increase in astral MT number and length yielding a ‘hairy’ spindle appearance during blue light exposure, and that these ectopic MTs rapidly disappear when blue light is switched off. (C) Color-coded maximum intensity projections of MTs in fixed metaphase spindles in the dark or after 10 min blue light exposure. Box plots show the comparison of astral MT length and number. Data from 4 independent experiments are indicated by different colors. Statistical analysis by unpaired Student’s t-test of the experimental mean. (D) π-EB1 H1299 cell expressing mScarlet-I-tagged Kif18B in the dark and during local blue light exposure of the right spindle pole (indicated by the blue box in the middle panel). Inset in the left panel shows KIF18B-mScarlet localization to growing astral MT ends at 2 s intervals. The graph shows the relative KIF18B-mScarlet fluorescence intensity over time comparing the non-illuminated and the blue light-exposed pole in n = 19 cells with the shaded areas indicating 95% confidence intervals and the solid blue line an exponential fit. Single channel images in B and D are shown with inverted contrast, and blue bars in all panels indicate time phases with blue light stimulation.

Article Snippet: The KIF18B coding sequence was amplified by PCR from 24xMoonTag-kif18b-24xPP7 (Addgene plasmid #128604 [ ] was a gift from Marvin Tanenbaum) and cloned into KpnI and BamHI sites of pCMV-CKAP5-mScarlet-I [ ] by Gibson Assembly thereby replacing the CKAP5 ORF with KIF18B. pEB1N-mApple-LZ-LOV2 was cloned by inserting the mApple and LZ-LOV2 coding sequences into the XhoI and BamHI sites of EB1N-mCherry-LZ-LOV2 [ ] by Gibson Assembly. mApple and LZ-LOV2 coding sequences were amplified using primers detailed in the key resource table. mCherry-Dynactin-C-18, representing the neuronal DCTN1 isoform, was obtained from the UCSF Michael Davidson plasmid collection.

Techniques: Comparison, Sequencing, Expressing, Fluorescence