Journal: Biology

Article Title: Activation of P2X7 Receptor Mediates the Abnormal Ovulation Induced by Chronic Restraint Stress and Chronic Cold Stress.

doi: 10.3390/biology13080620

Figure Lengend Snippet: Figure 3. The expression of P2X7R in ovaries of chronic stress mice and the influence of P2X7R signaling on the ovulation of mice. After establishing the chronic stress model, immunohistochemistry and Western blotting were used to examine the expression change of P2X7R in ovarian tissues. (A–C) show the effect of chronic restraint stress (CRS) and chronic cold stress (CCS) on the expression of P2X7R in ovarian tissues. Superovulation was used to assess the ovulation rate of mice. (D,E) show the effect of A-438079 HCL, an antagonist of P2X7R signaling, on the superovulation rate of CRS and CCS mice. (F) shows the effect of BzATP and A-438079 HCL on the superovulation rate of normal healthy mice. * indicates p < 0.05 and ** indicates p < 0.01. Scale bar = 80 µm. The uncropped western blot figures were presented in Figures S1 and S2.

Article Snippet: The sections were inactivated through 3% H2O2 for 10 min, incubated with 3% BSA to block nonspecific binding, and were incubated with primary P2X7R antibody (1:400 dilution, 28207-1-AP, Proteintech, Wuhan, China) overnight at 4 ◦C.

Techniques: Expressing, Immunohistochemistry, Western Blot

Journal: Biology

Article Title: Activation of P2X7 Receptor Mediates the Abnormal Ovulation Induced by Chronic Restraint Stress and Chronic Cold Stress.

doi: 10.3390/biology13080620

Figure Lengend Snippet: Figure 4. The influence of P2X7R signaling on cumulus expansion in vitro and the expression of ovulation-related factors. (A) shows the effect of BzATP, an agonist of P2X7R signaling, on cumulus expansion induced by EGF. EGF served as positive control of cumulus expansion. Real-time PCR was used to detect the change in expression of ovulation-related factors. (B) shows the effect of A-438079 HCL, an antagonist of P2X7R signaling, on the expression of ovulation-related factors in granulosa cells. We found that NPPC was down-regulated. (C) shows the effect of BzATP and A-438079 HCL on NPPC mRNA expression. (D) shows the effect of BzATP and A-438079 HCL on CNP protein level. * indicates p < 0.05 and ** indicates p < 0.01. Scale bar = 100 µm.

Article Snippet: The sections were inactivated through 3% H2O2 for 10 min, incubated with 3% BSA to block nonspecific binding, and were incubated with primary P2X7R antibody (1:400 dilution, 28207-1-AP, Proteintech, Wuhan, China) overnight at 4 ◦C.

Techniques: In Vitro, Expressing, Positive Control, Real-time Polymerase Chain Reaction

Journal: Biology

Article Title: Activation of P2X7 Receptor Mediates the Abnormal Ovulation Induced by Chronic Restraint Stress and Chronic Cold Stress.

doi: 10.3390/biology13080620

Figure Lengend Snippet: Figure 6. P2X7R signaling influenced the ovulation process through regulating NPPC expression and increased the ovarian fibrosis through regulating fibrosis-related factors, including IL-1β, TGF-β1 and MMP2, which may contribute to the abnormal ovulation of the chronic stress mouse model.

Article Snippet: The sections were inactivated through 3% H2O2 for 10 min, incubated with 3% BSA to block nonspecific binding, and were incubated with primary P2X7R antibody (1:400 dilution, 28207-1-AP, Proteintech, Wuhan, China) overnight at 4 ◦C.

Techniques: Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: Loss of alpha-kinase 1 contributes to the formation of congenital cataracts in mice

doi: 10.1186/s11658-025-00827-2

Figure Lengend Snippet: Loss of ALPK1 promoted the development of congenital cataracts (CCs) in mice. A Representative images and quantification of ALPK1 expression in the anterior lens epithelium determined by immunofluorescence (IF) staining in 8-week-old mice ( n = 6/group). *** p < 0.001, versus WT mice. B Fundus imaging and fluorescein fundus angiography (FFA) examination of 16-week-old mice. C Hematoxylin and eosin (HE) staining of retinal sections from 8-week-old WT and ALPK1 −/− mice. D Representative slit-lamp images of cataract morphology and the cataract incidence at the indicated ages in WT and ALPK1 −/− mice ( n = 15/group). OD, oculus dexter. OS, oculus sinister. * p < 0.05, ** p < 0.01, and ** *p < 0.001, versus age-matched WT mice

Article Snippet: These slides were incubated in 10% donkey serum to block nonspecific labeling, followed by an overnight incubation at 4 °C with primary antibodies against ALPK1 (1:100, ER65387, Huabio, Hangzhou, China), ZO-1 (1:500, 21,773–1-AP, Proteintech, Wuhan, China), C/EBP-homologous protein (CHOP, 1:500, GB11204, Servicebio), or aquaporin-0 (AQP0, 1:200, GB112258 , Servicebio).

Techniques: Expressing, Immunofluorescence, Staining, Imaging

Journal: Cellular & Molecular Biology Letters

Article Title: Loss of alpha-kinase 1 contributes to the formation of congenital cataracts in mice

doi: 10.1186/s11658-025-00827-2

Figure Lengend Snippet: Loss of ALPK1 disrupted the lens histomorphology and cellular morphology in mice with CCs. A Representative HE-stained lens sections from 8-week-old WT mice and ALPK1 −/− mice with CCs. The blue arrowheads indicate vacuole formation. B Representative transmission electron microscope (TEM) images of lens epithelial cells (LECs) and lens fiber cells (LFCs) in the normal lenses from wild-type (WT) mice and the cataract lenses from ALPK1 −/− mice with CCs. N, nucleus; ER, endoplasmic reticulum; Mi, mitochondria; Ig, intercellular gap. In LECs, the red, blue, yellow, orange, and green arrowheads indicate the dilated ER, dilated perinuclear space, chromatin condensation, swollen Mi, and dilated intercellular gap, respectively. In LFCs, the blue and red arrowheads indicate interlocking domains and membranous globules

Article Snippet: These slides were incubated in 10% donkey serum to block nonspecific labeling, followed by an overnight incubation at 4 °C with primary antibodies against ALPK1 (1:100, ER65387, Huabio, Hangzhou, China), ZO-1 (1:500, 21,773–1-AP, Proteintech, Wuhan, China), C/EBP-homologous protein (CHOP, 1:500, GB11204, Servicebio), or aquaporin-0 (AQP0, 1:200, GB112258 , Servicebio).

Techniques: Staining, Transmission Assay, Microscopy

Journal: Cellular & Molecular Biology Letters

Article Title: Loss of alpha-kinase 1 contributes to the formation of congenital cataracts in mice

doi: 10.1186/s11658-025-00827-2

Figure Lengend Snippet: Loss of ALPK1 promoted endoplasmic reticulum stress (ERS)-induced apoptosis in the LECs and abnormalities in the LFCs of 8-week-old mice. A Representative western blotting images and quantification of p-myosin Ia in WT mice and ALPK1 −/− mice with CCs, which were normalized to β -actin ( n = 3/group, each sample consisting of lens epithelial tissue from 5 mice, i.e. 10 lenses mixed together per sample). B Representative images of ZO-1 localization in the anterior and equatorial epithelia of WT and ALPK1 −/− mice. C Representative images and quantification of C/EBP-homologous protein (CHOP) immunoreactivity in the anterior and equatorial regions in WT and ALPK1 −/− mice ( n = 6/group). D Representative images and quantification of TUNEL-positive apoptotic cells in murine lenses ( n = 6 /group). E Representative images of AQP0 expression (red) in the cortical fibers of WT and ALPK1 −/− lenses. *** p < 0.001, versus WT group

Article Snippet: These slides were incubated in 10% donkey serum to block nonspecific labeling, followed by an overnight incubation at 4 °C with primary antibodies against ALPK1 (1:100, ER65387, Huabio, Hangzhou, China), ZO-1 (1:500, 21,773–1-AP, Proteintech, Wuhan, China), C/EBP-homologous protein (CHOP, 1:500, GB11204, Servicebio), or aquaporin-0 (AQP0, 1:200, GB112258 , Servicebio).

Techniques: Western Blot, TUNEL Assay, Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: Loss of alpha-kinase 1 contributes to the formation of congenital cataracts in mice

doi: 10.1186/s11658-025-00827-2

Figure Lengend Snippet: ALPK1 knockdown inhibited myosin Ia phosphorylation, promoted ERS-induced apoptosis, and impeded differentiation of primary LECs. The protein levels of ALPK1, p-myosin Ia, CHOP, CK19, and AQP0, as well as apoptosis levels, were determined in primary mouse LECs treated with medium only (control), sgRNA-NC or sgRNA-ALPK1 lentivirus, respectively ( n = 4/group). Besides p-myosin Ia, their relative levels were calculated using FI and expressed as positive ratio relative to control. A Representative images and quantification of ALPK1 immunoreactivity. B Representative western blotting images and quantification of p-myosin Ia protein. Protein levels were normalized to β -actin and expressed as fold changes relative to control. C Representative images and quantification of CHOP immunoreactivity. D Representative images and quantification of TUNEL-positive apoptotic cells. E Representative images of the positive immunoreactivity for CK19 or AQP0. The relative FI ratio of CK19/AQP0 was calculated and expressed as fold changes relative to control. ** p < 0.01, *** p < 0.001, versus sgRNA-NC group

Article Snippet: These slides were incubated in 10% donkey serum to block nonspecific labeling, followed by an overnight incubation at 4 °C with primary antibodies against ALPK1 (1:100, ER65387, Huabio, Hangzhou, China), ZO-1 (1:500, 21,773–1-AP, Proteintech, Wuhan, China), C/EBP-homologous protein (CHOP, 1:500, GB11204, Servicebio), or aquaporin-0 (AQP0, 1:200, GB112258 , Servicebio).

Techniques: Knockdown, Phospho-proteomics, Control, Western Blot, TUNEL Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Loss of alpha-kinase 1 contributes to the formation of congenital cataracts in mice

doi: 10.1186/s11658-025-00827-2

Figure Lengend Snippet: Lentiviral-mediated ALPK1 restoration in the lens prevented cataract formation in ALPK1 −/− mice. A IF staining of LV-ALPK1-transduced primary mouse LECs. B Representative IF images and quantification of ALPK1 expression in the anterior lens epithelia in 8-week-old ALPK1 −/− mice treated with LV-GFP or LV-ALPK1 ( n = 12/group). *** p < 0.001, versus ALPK1 −/− mice treated with LV-GFP. C Representative slit-lamp images of cataract morphology and the cataract incidence at the indicated ages in the ALPK1 −/− mice treated with LV-GFP or LV-ALPK1 ( n = 12/group). OD, oculus dexter; OS, oculus sinister. * p < 0.05, ** p < 0.01, versus age-matched mice in LV-ALPK1 treated ALPK1 −/− mice. D Representative HE-stained lens sections from 8-week-old ALPK1 −/− mice treated with LV-GFP or LV-ALPK1. The blue arrowheads indicate the vacuole formation in murine lenses

Article Snippet: These slides were incubated in 10% donkey serum to block nonspecific labeling, followed by an overnight incubation at 4 °C with primary antibodies against ALPK1 (1:100, ER65387, Huabio, Hangzhou, China), ZO-1 (1:500, 21,773–1-AP, Proteintech, Wuhan, China), C/EBP-homologous protein (CHOP, 1:500, GB11204, Servicebio), or aquaporin-0 (AQP0, 1:200, GB112258 , Servicebio).

Techniques: Staining, Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: Loss of alpha-kinase 1 contributes to the formation of congenital cataracts in mice

doi: 10.1186/s11658-025-00827-2

Figure Lengend Snippet: LV-ALPK1-induced ALPK1 expression attenuated lens pathological alteration in ALPK1 −/− mice. Two-week-old ALPK1 −/− mice without cataract received an injection of LV-GFP or LV-ALPK1 into the anterior chamber of the eye. After 6 weeks, the lenses of 8-week-old mice were collected for analysis. A Representative images of ZO-1 localization in the LECs. B Representative images and quantitative analysis of CHOP immunoreactivity in the anterior and equatorial regions. n = 12/group. *** p < 0.001, versus LV-GFP treated ALPK1 −/− mice. C Representative images and quantification of TUNEL-positive apoptotic cells in the lenses. n = 12/group. *** p < 0.001, versus LV-GFP-treated ALPK1 −/− mice. D Representative images of AQP0 expression (red) in the lens cortical fibers

Article Snippet: These slides were incubated in 10% donkey serum to block nonspecific labeling, followed by an overnight incubation at 4 °C with primary antibodies against ALPK1 (1:100, ER65387, Huabio, Hangzhou, China), ZO-1 (1:500, 21,773–1-AP, Proteintech, Wuhan, China), C/EBP-homologous protein (CHOP, 1:500, GB11204, Servicebio), or aquaporin-0 (AQP0, 1:200, GB112258 , Servicebio).

Techniques: Expressing, Injection, TUNEL Assay