Fig 2: Myosin 1f is upregulated after the mouse model of retinal detachment.A, B The expression of myosin 1f at day 1, day 3, day 7 on western blot, it reached to peak at day 3 (A). The expression value is calculated by the optical density ratio of myosin 1f and GAPDH (B). C The expression of myosin 1f at day 1, day 3, day 7 on qPCR. D The count of F4/80 and myosin 1f-positive cells on eye sections under every scope (×40). E The count of IBA1 and myosin 1f-positive cells on eye sections under every scope (×40). F The Representative image of eye sections stained for F4/80 (green), myosin 1f (red), and dapi (blue). The eyeballs were taken down on day 3 after RD. G Representative image of eye sections stained for IBA1 (green), myosin 1f (red), and dapi (blue) (day 3). H Eye sections stained for GFAP (green), myosin 1f (red), and dapi (blue) (day 3). I Eye sections stained for Tuj1 (green), myosin 1f (red), and dapi (blue) (day 3). J Eye sections stained for Opsin (green), myosin 1f (red), and dapi (blue) (day 3). K Myosin 1f is also upregulated in rd1 mouse day 7 after birth, quantification value is calculated by the optical density ratio of myosin 1f and GAPDH. L Myosin 1f is also upregulated in the light-injured retina (day 5), quantification value is calculated by the optical density ratio of myosin 1f and GAPDH. M Retinal sections of rd1 mice (day 7 after birth) stained for F4/80 (green), myosin 1f (red), and dapi (blue). N Retinal sections of the light-injured retina (day 5) stained for F4/80 (green), myosin 1f (red), and dapi (blue). Data were presented as mean ± SEM, unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: The overexpression of DsbA-L aggravated I/R-induced kidney damage was attenuated in PT-VDAC1-KO mice.The PT-VDAC1-KO and PT-DsbA-L-WT mice were treated with or without DsbA-L plasmid and then established an IR model. (a)BUN. (b)Serum creatinine. (c) Hematoxylin and eosin staining. (d) Representative sections of TUNEL-positive cells. (e) Tubular damage score. (f) The number of TUNEL-positive cells. (g) The immunoblots analysis the expression of cleaved caspase3, VDAC1, and DsbA-L as well as the expression of BAX and Cyt-c in mitochondrial and cytosolic fractions. ß-tubulin and GAPDH was used as whole lysate or cytosolic loading control, respectively. COX IV was used as mitochondria loading control. (h–n) Analysis of the gray scale image between them. Original magnification x 400. Scar Bar:100µM. Each experiment(c–e) was repeated 6 times independently with similar results. One-way ANOVA was used for the Multiple group comparisons (a, b and f–n). # p<0.05: versus saline group, sham group, or MCD group. # P<0.05 versus sham group. ^ P<0.05 versus IR group.* P<0.05 versus IR group.

Fig 4: Plk4 binds Cdc6 in S phase and phosphorylates Cdc6.(a) Endogenous Cdc6 interacts with Plk4 in cells. HEK293 total cell extract was immunoprecipitated with a Cdc6 antibody, and probed with Cdc6 and Plk4 antibodies. (b) Plk4 directly binds Cdc6 in vitro. Sepharose beads coupled with purified GST or GST-Plk4 were incubated with purified His-Cdc6 and analysed using His antibody. The loadings of indicated proteins are shown by Coomassie blue staining. (c) The Plk4 kinase-dead mutant K41M does not bind Cdc6 in cells. HEK293 cells were co-transfected with Myc-Cdc6 and GFP-Plk4 WT or K41M mutant. Total cell extracts were immunoprecipitated with GFP antibody, and probed with Myc and GFP antibodies. (d) Cdc6 interacts with Plk4 during S phase. HEK293 cells were co-transfected with GFP-Plk4 and Myc-Cdc6 and synchronized at the G1/S transition. The cells were then released for the indicated time period, collected for immunoprecipitation using GFP antibody, and analysed by western blotting using Myc, GFP, cyclin B and GAPDH antibodies. (e) N-terminus of Cdc6, but not C-terminus of Cdc6, interacts with Plk4. GFP, GFP-tagged Cdc6 WT or Cdc6 truncate was immunoprecipitated with GFP antibody from total cell extract of HEK293 cells co-transfected with Myc-Plk4. Immunoprecipitated proteins were analysed using Myc and GFP antibodies. (f) Cdc6 is phosphorylated on S30 and T527 in cells. HEK293 cells were transfected with GFP-tagged Cdc6 or Cdc6 mutants, and the cell lysates were probed with GFP antibody using Phos-Tag acrylamide assay (upper panel). (g) Plk4 phosphorylates Cdc6 on S30 and T527 in vitro. Purified GST-Plk4 was incubated with purified His-tagged Cdc6 or Cdc6 mutant proteins in the presence of [?-32P]-ATP, followed by autoradiography. Coomassie blue staining shows the loading of indicated proteins. Asterisk indicates the phosphorylated Cdc6. (h) Quantitation of (g) for Cdc6 WT, S30A, T527A or 2A phosphorylation from four independent experiments. The relative intensity of the phosphorylation signal of each protein was normalized to its protein level by ImageJ. The phosphorylation signal intensity of Cdc6 WT was arbitrarily set at an intensity of 1.0. The statistical data in h is presented as means±s.d. ***P<0.001 (Student's t-test).

Fig 5: Shikonin induced autophagy in RCC cells. (A) ACHN (right panel) and Caki-1 (left panel) cells were treated with 0 or 6 µM shikonin for 24 h and subjected to acridine orange staining and the images were captured under a fluorescence microscope. (B) ACHN and Caki-1 cells were treated with 0, 4, 6, and 8 µM shikonin with or without NAC pretreatment. The cell lysates were analyzed by Western blot using LC3B and p62 antibodies. GAPDH was used as the loading control. Data represent means ± standard deviation from at least three independent experiences. ** p < 0.01 and *** p < 0.001 compared with the control group. # p< 0.05, ## p < 0.01 and ### p < 0.001 compared with the NAC pretreated group. GAPDH was used as the internal control. (C) ACHN and Caki-1 cells were treated with 6 µM shikonin in the presence or absence of LC3B-II siRNA (siLC3B) for 24 h. The cell lysates were analyzed by Western blot using LC3B antibody. (D) ACHN and Caki-1 cells were treated with 6 µM shikonin in the presence or absence of siLC3B for 24 h. Cell viability was measured by MTT assay. Data represent means ± standard deviation from at least three independent experiences. ** p < 0.01 and *** p < 0.001 compared with the control group. # p < 0.05 compared with the siLC3B group.