Journal of Jinan University Natural Science & Medicine Edition

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Human Krt5 promotes cell filopodia formation and wound healing in zebrafish fibroblasts

CHEN Kaixing, YU Feifei, WU Wenhui, YAN Jizhou

Journal of Jinan University Natural Science & Medicine Edition, 2023, 44(3): 227-237. DOI: 10.11778/j.jdxb.20220366

处理方法	基因	评分	覆盖率/%	肽段数目¹⁾	肽段谱图数	分子量/kD
6×His-Krt5处理用Ni-TAT纯化	Myh9b	126.13	42.17	70	95	197.53
	Actn1	99.43	54.88	60	109	104.21
	Myh10	84.15	32.18	53	66	231.16
	Cad	59.06	16.27	27	30	247.36
	Golga2	47.18	26.80	26	32	120.77
	Hnrnpub	46.20	29.40	26	43	88.55
	Rpn1	44.13	41.47	23	32	67.66
	Hspa5	41.41	38.00	20	27	71.99
6×His-Krt5+Noggin-Fc处理用Ni-TAT纯化	Myh9b	102.18	34.92	52	66	197.53
	Actn1	83.75	49.56	50	88	104.21
	Myh10	67.03	25.25	38	43	231.16
	Cad	63.06	16.41	32	38	247.36
	Hnrnpub	43.45	25.13	21	28	88.55
	Hspa5	39.36	36.15	19	28	71.99
6×His-Krt5+Noggin-Fc处理用Protein A/G纯化	Myh9b	21.81	8.95	10	11	197.53
	Actbb	15.00	30.67	8	12	41.75
	Myh10	8.54	3.72	6	6	231.16
Noggin-Fc处理用Protein A/G纯化	Myh9b	98.03	29.51	49	74	197.53
	Myh10	67.78	23.19	41	46	231.16
	Actbb	33.65	47.20	19	52	41.75
	Hspa8	28.56	26.70	14	15	71.21

Table 3 Affinity purified 6×His-Krt5 and/or Noggin-Fc protein complexes identified by mass spectrometry

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Table 1 Primer sequences required for in vitro synthesis of Krt5 mRNA
Figure 1 Identification of target gene prokaryotic expression A: PCR identification of prokaryotic expression plasmid. The lanes were sequentially DNA Marker, plasmid PCR products without inserting the target gene, and recombinant plasmid PCR products containing IL-1β, IL-4, IL-6, and IL-10 protein coding regions; B: SDS-PAGE identification of His tag target protein. Lane 1 was the uninduced bacterial liquid, lane 2 was the induced bacterial liquid, and lane 3 was the purified protein. The molecular weights of His labeled proteins of IL-1β, IL-4, IL-6, and IL-10 were 45.07, 31.82, 38.04 and 34.84 kD.
Figure 2 Krt5 mRNA was synthesized in vitro and expressed in transfected PAC2 cells A: Electrophoretic Results of Intermediate Products of Krt5 mRNA Synthesized in Vitro. Lane 1 is the PCR product of Krt5 gene, lane 2 is the PCR product with added UTR sequence, and lane 3 is the PCR product with poly(T) tail; B: Western blot detection of Krt5 expression in PAC2 cells.
Figure 3 Overexpressed Krt5 promotes the formation of filopodia in PAC2 cells The green fluorescence shows the Alexa Fluor 488-Phalloidin stained F-actin, the red fluorescence shows the location of Krt5 in the cell, the blue fluorescence shows the DAPI stained nucleus, and the white arrow indicates the filopodia.
Figure 4 Effects of overexpression of Krt5 on the migration ability of PAC2 cells A: Effects of overexpression of Krt5 at different mass concentration on the migration ability of PAC2 cells; B: Comparison of the corresponding scratch area and healing rate at each time point. 1) Compared with the Control group, P<0.05.
Table 2 FPKM values of differentially expressed genes in KEGG enrichment analysis treated by Krt5 and macrophage polarization factor
Figure 5 Relative expression levels of differential genes in KEGG pathway after Krt5 overexpression A: TGF-β receptor gene expression changes after Krt5 overexpression; B: KEGG enriched differential gene expression changes after Krt5 overexpression; C: Interleukin-receptor gene expression changes after Krt5 overexpression. 1)Compared with group A, P<0.01.

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