Journal: Scientific Reports

Article Title: Autism-associated mutation in Hevin/Sparcl1 induces endoplasmic reticulum stress through structural instability

doi: 10.1038/s41598-022-15784-5

Figure Lengend Snippet: Hevin mutants lacking the EF-hand motif activate the UPR signaling caused by abnormal trafficking. ( A ) Immunoblotting of Hevin WT and mutants (ΔEF and N11). Neuro-2a cells were transfected for 48 h with expression vectors for Hevin WT or mutants (ΔEF or N11). The cell lysates and conditioned medium were subjected to immunoblot analysis using anti-Hevin and Tubulin antibodies. ( B ) Immunostaining of Hevin and mutants (ΔEF and N11). HeLa cells were transfected for 24 h with expression vectors for Hevin and mCherry-ER and subjected to immunocytochemistry. Scale bar: 10 μm. ( C ) Quantification of Pearson’s correlation coefficient as the degree of colocalization in the panel B. n = 20 cells, mean ± standard error of the mean (SEM), ** p < 0.01, *** p < 0.001 versus Hevin WT, one-way analysis of variance (ANOVA) Dunnett’s test. ( D , E ) Total RNAs isolated from the Hevin-overexpressed Neuro-2a cells were conducted with qPCR analysis for measurement of Bip and Chop mRNAs. 5S rRNAs were used for normalization. n = 5; mean ± SEM; * p < 0.05, ** p < 0.01 versus Hevin WT, one-way ANOVA Dunnett’s test calculated using the ΔCt value. ( F ) Immunoblotting of BIP, Hevin, and Tubulin. HEK293T cells were transfected for 72 h with expression vectors for Hevin WT or mutants (ΔEF or N11). Cells were stimulated with 500 nM Thapsigarging (Tg) for 24 h. The cells were then subjected to immunoblot analysis using anti-BIP, Hevin and Tubulin antibodies.

Article Snippet: For immunoblotting goat anti-Hevin (1:1000, R&D Systems, Cat# AF2836), mouse anti-Tubulin (1:1000, Sigma, DM1A), rabbit anti-BIP (1:1000, Cell Signaling, CB0B12), and mouse anti-GST (1:500, Santa Cruz, B14) antibodies were used as primary antibodies.

Techniques: Western Blot, Transfection, Expressing, Immunostaining, Immunocytochemistry, Isolation

Journal: Scientific Reports

Article Title: Autism-associated mutation in Hevin/Sparcl1 induces endoplasmic reticulum stress through structural instability

doi: 10.1038/s41598-022-15784-5

Figure Lengend Snippet: ASD-associated W647R mutant of Hevin activates the UPR signaling. ( A ) Location of ASD-associated Hevin mutations. ( B ) Amino acid of the EF-hand motif in each species. The human Trp 647 is highly conserved. Cyan indicates the EF-hand motif, Magenta indicates non-conserved amino acid. ( C ) Immunoblotting of Hevin WT and mW633R mutant. Neuro-2a cells were transfected with expression vectors and collected at the indicated times. The upper panel indicates the experimental schedule. The transfection efficiencies were confirmed by observing co-expressed EGFP fluorescence. The cell lysates and conditioned medium were subjected to immunoblot analysis using anti-Hevin and Tubulin antibodies. The secretion rate of the Hevin WR mutant was delayed more than that of WT. CM; conditioned medium, TCL: total cell lysate ( D ) Immunostaining of Hevin WT and mW633R mutant. HeLa cells were transfected for 24 h with expression vectors for Hevin and mCherry-ER and subjected to immunocytochemistry. Scale bar: 10 μm. ( E ) Quantification of Pearson’s correlation coefficient as the degree of colocalization in the panel D. n = 15 cells, mean ± SEM, *** p < 0.001 by Student’s t- test. ( F ) Immunostaining of Hevin and mW633R mutant. HeLa cells were transfected for 24 h with expression vectors for Hevin and subjected to immunocytochemistry. Scale bar: 10 μm. ( G ) Quantification of Manders’ coefficient as the degree of colocalization of Hevin with GM130 in the panel F. n = 20 cells, mean ± SEM, *** p < 0.001 by Student’s t- test. ( H , I ) Total RNAs isolated from the Hevin-overexpressed Neuro-2a cells were conducted with qPCR analysis for measurement of Bip and Chop mRNAs. 5S rRNA were used for normalization. n = 5; ** p < 0.01, *** p < 0.001 versus Hevin WT, one-way ANOVA Dunnett’s test calculated p -value using the ΔCt value. ( J ) Immunoblotting of BIP, Hevin, and Tubulin. Neuro-2a cells were transfected for 72 h with expression vectors for Hevin WT or mW633R mutants. The cell lysates were then subjected to immunoblot analysis using anti-BIP, Hevin, and Tubulin antibodies. ( K ) Schematic structure of GST-Hevin. ( L ) Pull down of GST-Hevin and endogenous BIP in HEK293T cells. GST-Hevin was precipitated with Glutathione Sepharose beads and immunoblotted with anti-GST, BIP, and Tubulin antibodies.

Article Snippet: For immunoblotting goat anti-Hevin (1:1000, R&D Systems, Cat# AF2836), mouse anti-Tubulin (1:1000, Sigma, DM1A), rabbit anti-BIP (1:1000, Cell Signaling, CB0B12), and mouse anti-GST (1:500, Santa Cruz, B14) antibodies were used as primary antibodies.

Techniques: Mutagenesis, Western Blot, Transfection, Expressing, Fluorescence, Immunostaining, Immunocytochemistry, Isolation