1. Volume 25, Issue 5, Pages 634-643.e4 (May 2018)
A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome 
Kohei Omachi, Misato Kamura, Keisuke Teramoto, Haruka Kojima, Tsubasa Yokota, Shota Kaseda, Jun Kuwazuru, Ryosuke Fukuda, Kosuke Koyama, Shingo Matsuyama, Keishi Motomura, Tsuyoshi Shuto, Mary Ann Suico, Hirofumi Kai 
Cell Chemical Biology 
Volume 25, Issue 5, Pages e4 (May 2018)
DOI: /j.chembiol
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2. Cell Chemical Biology 2018 25, 634-643. e4DOI: (10. 1016/j. chembiol
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3. Figure 1
Intracellular α5(IV) Monomers Are Stable Despite Harboring a Mutation
(A) Immunoblots of Myc-tagged WT and mutant α5(IV) monomer in HEK293T cells treated with cycloheximide for chase experiments.
(B) Quantification of intracellular α5(IV) expression normalized to vinculin in (A). Data are presented as percentage of the amount detected at 0 hr. Graphs indicate mean ± SE (n = 3).
(C) Immunoblots of Myc-tagged WT and mutant α5(IV) monomer in HEK293T cells treated with the indicated reagents for 24 hr.
(D) Quantification of intracellular α5(IV) expression normalized to vinculin in (C). Data are presented as percentage of the amount detected in the Mock group. Error bars indicate mean ± SE (n = 3). *p < 0.05, **p < 0.01 versus Mock (Tukey-Kramer test).
(E) Intracellular expression of Myc-tagged WT and C1567R α5(IV) monomers in HEK293T cells co-transfected with SAR1 GTP.
(F) Quantification of intracellular α5(IV) expression normalized to vinculin in (E). Data are presented as percentage of the amount detected in the Mock group. Error bars indicate the mean ± SE (n = 3). *p < 0.05, **p < 0.01 versus Mock (Student's t test).
Blots in (A), (C), and (E) were probed with anti-Myc or anti-vinculin antibodies. See also Figure S1.
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4. Figure 2
WT and Mutant α5(IV) Chains Are Similarly Regulated by Typical ER Chaperones
(A and B) Blots of intracellular and extracellular Myc-tagged α5(IV) monomer expression in HEK293T cells co-transfected with the indicated ER chaperones (A) or siRNA (B). O.E., overexpression. Blots were probed with antibodies indicated at the right side of the panels. See also Figure S2.
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5. Figure 3
Establishment of a Method for Assessing α345(IV) Trimer Formation that Reflected Established Features of Collagen Regulation
(A) The scheme of split NanoLuc-based α345 (IV) trimer assay.
(B–J) Luminescence was measured in the media (extracellular) from HEK293T cells transfected with: (B) C-terminal LgBiT- or SmBiT-α3/α4/α5(IV) in the indicated combination. (C) C-terminal or (D) N-terminal LgBiT-α5(IV), SmBiT-α3(IV), and COL4A4-3FLAG in the indicated combinations. Left panels: scheme of α(IV)-tagged constructs. S.S., signal sequence. (E) C-terminal LgBiT-α5(IV), SmBiT-α3(IV), and empty vector or serial amounts of expression vectors encoding COL4A4-3FLAG. (F and G) C-terminal LgBiT-α5(IV), SmBiT-α3(IV), COL4A4-3FLAG, and empty vector or expression vectors encoding COL4A3-HA (F) or COL4A5-Myc (G). (H) C-terminal LgBiT-WT, -ΔCOL, or -ΔNC1 α5(IV), SmBiT-α3(IV), and COL4A4-3FLAG. Left panel: scheme of α(IV)-tagged constructs. (I) C-terminal LgBiT-α5(IV), SmBiT-α3(IV), and COL4A4-3FLAG treated with 200 μM ascorbic acid for 24 hr. (J) Single α-chain-expressing cells (α3, α4, α5), co-cultured single α-chain-expressing cells (α3+α4+α5), or triple α-chain-expressing cells (α345).
In (B) to (J), error bars indicate mean ± SE (n = 4). **p < 0.01 versus α3, α5, and α35 (C and D); **p < 0.01 versus WT (H); **p < 0.01 versus α3, α4, α5 and α3+α4+α5 (J) (B–E, J, Tukey-Kramer; F–I, Student's t test). See also Figure S3.
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6. Figure 4
The Split NanoLuc α345(IV) Trimer Assay Enabled the Assessment of Clinically Reported α5(IV) Mutants
(A and B) Luminescence was measured in the media from cells expressing α3, α4, and C-terminal-tagged (A) or N-terminal-tagged (B) WT or the indicated mutant α5(IV). Error bars indicate the mean ± SE (n = 4). **p < 0.01 versus WT (Tukey-Kramer test). See also Figure S4.
(C and D) Scatterplot of the intracellular/secreted RLU ratio from cells expressing the WT or mutant α5 chain. Solid line: y = x; dotted line: y = x + 30, y = x − 30. White square, WT; black circles, intracellular formation-dependent defect; gray circles, secretion-dependent defect.
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7. Figure 5
Some Chemical Chaperones but Not Typical ER Chaperones Were Able to Correct the Trimer Formation of Mutant α5(IV)
(A and B) Luminescence was measured in media from cells expressing C-terminal-tagged α34 and WT or G869R α5 co-transfected with the indicated ER chaperone expression vectors (A) or siRNA (B). Error bars indicate mean ± SE (n = 4). ##p < 0.01 versus Mock WT α5; *p < 0.05, **p < 0.01 versus Mock G869R α5 (Tukey-Kramer test).
(C) Validity of the 96-well format of the C-terminal split NanoLuc-based α345 trimer assay.
(D–F) Luminescence was measured in media from cells expressing C-terminal-tagged α34 and G869R α5 treated with the indicated chemical chaperones (D), or serial concentrations of mannitol (E) and TMAO (F). Data are presented as mean ± SE (n = 4). *p < 0.05, **p < 0.01 versus Mock G869R α5 (Tukey-Kramer test).
(G–J) Luminescence was measured in media from cells expressing C-terminal-tagged α34 and G1107R (G), G1244D (H), G1143D (I), and C1567R (J) α5(IV) treated with the indicated chemical chaperones. Data are presented as mean ± SE (n = 4). ##p < 0.01 versus Mock WT α5; **p < 0.01 versus Mock mutant α5 (Tukey-Kramer test).
See also Figure S5.
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