Journal: Gut Microbes

Article Title: Intestinal epithelial pH-sensing receptor GPR65 maintains mucosal homeostasis via regulating antimicrobial defense and restrains gut inflammation in inflammatory bowel disease

doi: 10.1080/19490976.2023.2257269

Figure Lengend Snippet: GPR65 deficiency drives distinct IEC transcriptional programs and compromises the downstream STAT3 signaling. colonic IECs were isolated from Gpr65 ΔIEC mice and Gpr65 fl/fl littermates for RNA sequencing analysis. (a, b) GSEA analysis. (c) Heatmap of DEGs related to IEC antimicrobial gene profiles. (d) a schematic overview of the AMP induction assay by systemic injection with rmIL-17A or rmIL-22. (e, f) qPCR analysis of relative mRNA expression of Reg3g and Reg3b . (g) representative microscopical photographs of colonoids from Gpr65 ΔIEC and littermate Gpr65 fl/fl mice on day 10 of culture. Original magnification: × 40. (h, i) the colonoids from Gpr65 ΔIEC and Gpr65 fl/fl mice were stimulated with or without rmIL-22 (100 ng/mL) for 24 h. The relative mRNA expression of Reg3g and Reg3b was detected by qPCR analysis. (j) GSEA analysis for Hallmark_JAK_STAT3_Signaling. (k) STAT3-focused interaction network of genes downregulated by GPR65 signaling. (l) colonic IECs were isolated from Gpr65 ΔIEC mice and Gpr65 fl/fl littermates, and stimulated with rmIL-22 (100 ng/mL) for 15 min. The protein levels of p-STAT3 and STAT3 were determined by immunoblotting analysis. (m) immunoblotting analysis of p-STAT3 and STAT3 in the colonic IECs from the indicated groups of mice. (n) immunoblotting analysis of p-Erk1/2, Erk1/2, p-mTOR, mTOR, and β-actin in the colonic IECs from the indicated groups of mice. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are representative of three independent experiments.

Article Snippet: Primary antibodies against GAPDH (Cat: 5174S), STAT3 (Cat: 4904T), phosphorylated (p)-STAT3 (Cat: 4113S), Erk1/2 (Cat: 9102S), p-Erk1/2 (Cat:4370T), mTOR (Cat: 2983T), p-mTOR (Cat: 2971S), and HRP-linked secondary antibodies against rabbit (Cat: 7074) and mouse (Cat: 7076) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Isolation, RNA Sequencing Assay, Injection, Expressing, Western Blot

Journal: Renal Failure

Article Title: Butylphthalide improves brain damage induced by renal ischemia-reperfusion injury rats through Nrf2/HO-1 and NOD2/MAPK/NF-κB pathways

doi: 10.1080/0886022X.2023.2259234

Figure Lengend Snippet: The antibody Information of Western blot.

Article Snippet: p-ERK1/2 Antibody , Cell Signaling Technology , 4370T , 1:1000.

Techniques: Western Blot

Journal: Renal Failure

Article Title: Butylphthalide improves brain damage induced by renal ischemia-reperfusion injury rats through Nrf2/HO-1 and NOD2/MAPK/NF-κB pathways

doi: 10.1080/0886022X.2023.2259234

Figure Lengend Snippet: NBP activates Nrf2/HO-1 and inhibits NOD2/MAPK/NF-κB signaling pathway in the brain tissue of renal IR rats. (A-D) Western blot was used to detect the protein expression levels of Nrf2, HO-1, NLRP3, Caspase-1, NOD2, p-ERK1/2, p-JNK, p-p38, p-P65 and p-IkBa in the rat brain ( n = 3). Data were presented as the mean ± standard deviation, ▲ p < 0.05, ▲▲ p < 0.01.vs. sham group; ↔ p < 0.05, ↔↔ p < 0.01.vs. I/R group. Nrf2: NF-E2-related factor 2; HO-1: Heme oxygenase-1.

Article Snippet: p-ERK1/2 Antibody , Cell Signaling Technology , 4370T , 1:1000.

Techniques: Western Blot, Expressing, Standard Deviation

Journal: Renal Failure

Article Title: Butylphthalide improves brain damage induced by renal ischemia-reperfusion injury rats through Nrf2/HO-1 and NOD2/MAPK/NF-κB pathways

doi: 10.1080/0886022X.2023.2259234

Figure Lengend Snippet: NBP Protects BMVECs by activating the Nrf2/HO-1 and inhibiting NOD2/MAPK/NF-κB signaling pathway. (A-D) Western blot was used to detect the protein expression levels of Nrf2, HO-1, NLRP3, Caspase-1, NOD2, p-ERK1/2, p-JNK, p-p38, p-P65 and p-IkBα in the total BMVEC protein ( n = 3). ▲ p < 0.05, ▲▲ p < 0.01.vs. OGD group; ↔ p < 0.05, ↔↔ p < 0.01.vs. 10 μM NBP + OGD group; # p < 0.05, ## p < 0.01.vs. 10 μM NBP + MDP + OGD group.

Article Snippet: p-ERK1/2 Antibody , Cell Signaling Technology , 4370T , 1:1000.

Techniques: Western Blot, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Temporospatial inhibition of Erk signaling is required for lymphatic valve formation

doi: 10.1038/s41392-023-01571-9

Figure Lengend Snippet: rasa1 regulates lymphatic valve specification through inhibiting Erk signaling. a p-Erk1/2 immunostaining reveals increased Erk signal in FCLV LECs in rasa1a −/− ;rasa1b −/− and rasa1a(-3) −/− ; rasa1b −/− mutant embryos at 77 hpf. Solid lines, FCLV; yellow dashed lines, other tissues with p-Erk1/2 signals. Arrows indicate the obvious p-Erk1/2 signals in FCLV. Right, enlargement of the boxed regions in the left panels. Scale bars, 20 μm (left) or 10 μm (right). b Relative p-Erk1/2 intensity compared to lyve1b:DsRed2 expression in FCLV in wild-type and mutant embryos. Unpaired two-tailed t test (at least 3 independent experiments; Wild-type n = 7; rasa1a −/− ; rasa1b −/− n = 5; rasa1a(-3) −/− ; rasa1b −/− n = 8). c Selumetinib treatment from 2–4 dpf restored the valve structure in rasa1a(-3) −/− ; rasa1b −/− labeled with gata2a:EGFP and also induced ectopic gata2a:EGFP expression in the FLV. Arrowheads, LVs; arrows, FCLV-PHS LVVs; yellow arrows, RFLS-CCV LVVs; yellow arrowheads, ectopic gata2a:EGFP positive cells; asterisks, OLVs. The numbers of embryos with exhibited valve structures are shown. Scale bars, 50 μm. d Selumetinib treatment restores the LV and LVV formation in rasa1a(-3) −/− ;rasa1b −/− mutants at 4 dpf. Prox1a immunostaining was used to label the valve-forming LECs. Prox1a expressions were also presented in Fire LUT (Fiji). Arrowheads, LVs; arrows, FCLV-PHS LVVs. N = 3 independent experiments. Scale bars, 20 μm. e Statistical analysis of the valve-forming LECs in siblings with DMSO ( n = 11), rasa1a(-3) −/− ;rasa1b −/− mutant (Mt) with DMSO ( n = 10), and Mt with 100 μM selumetinib ( n = 20) in ( d ). Unpaired two-tailed t test. All images are anterior to the left, dorsal upward

Article Snippet: Mouse Erk 1/2 antibody (1/2000; Santa Cruz, 514302), rabbit p-Erk1/2 antibody (1/5000; CST, 4370), and rabbit Actb antibody (1/100000; Abclonal, AC026) were used for primary incubation.

Techniques: Immunostaining, Mutagenesis, Expressing, Two Tailed Test, Labeling

Journal: Signal Transduction and Targeted Therapy

Article Title: Temporospatial inhibition of Erk signaling is required for lymphatic valve formation

doi: 10.1038/s41392-023-01571-9

Figure Lengend Snippet: Inhibition of MAPK/Erk signaling leads to valve hyperplasia. a erk1 −/− ;erk2 −/− double mutants develop obvious edema at 5 dpf. Arrowheads indicate the edema in the heart and gut regions. Scale bar, 200 μm. b Statistical data for pericardial edema in the offspring of erk1 -/+ ; erk2 −/− mutants intercrossing at 5 dpf. Two-sided Fisher’s exact test. c erk1 −/− ;erk2 −/− double mutants show defects in both lymphatic vessel and valve formation. In erk1 −/− ;erk2 −/− double mutants, two types of valve phenotypes are observed, which are summarized in the schematic drawings on the right. Asterisks, anterior LFLs; arrowheads, lymphatic valves; arrows, FCLV-PHS LVVs. Scale bars, 50 μm. d At 5 dpf, Prox1a immunostaining reveals an increase of valve cells in the FCLV-PHS LVV and type II FCLV-LV in erk1 −/− ;erk2 −/− double mutants. The number of mutants with type II valve is indicated. Solid lines indicate FCLV and dashed lines indicate LFL. Scale bars, 50 μm. e Statistical analysis of the valve-forming LECs with high Prox1a expression in erk1 +/+ ;erk2 −/− ( n = 7) siblings or erk1 −/− ;erk2 −/− ( n = 16) mutants at 5 dpf in ( d ). Unpaired two-tailed t test (LV for erk1 +/+ ;erk2 −/− n = 7; LV for erk1 −/− ;erk2 −/− n = 16; FCLV-LVV for erk1 +/+ ;erk2 −/− n = 5; FCLV-LVV for erk1 −/− ;erk2 −/− n = 11). f Detection of the valve-forming LECs by Prox1a and gata2a:EGFP immunostaining in embryos treated with different doses of selumetinib from 2 to 4 dpf. Brackets indicate the LV structures. Two different types of LVs are observed. The numbers of embryos with the Prox1a expression pattern (magenta) in LVs are indicated. Scale bars, 20 μm. g Statistical analysis of LV cells after selumetinib treatment in ( f ). Unpaired two-tailed t test, (DMSO n = 7; 10 n = 7; 50 n = 6; 100 μM n = 3 and 5). h Statistical analysis of FCLV cells, excluding the valve-forming LECs with high Prox1a expression in ( f ). Unpaired two-tailed t test, (DMSO n = 7; 10 n = 6; 50 n = 7; 100 μM n = 8). i Detection of FCLV-PHS LVV formation in embryos treated with different doses of selumetinib from 2 to 4 dpf. The numbers of embryos with the Prox1a expression pattern (magenta) are indicated. Scale bars, 20 μm. j Statistical analysis of FCLV-PHS LVV cells in ( i ). Unpaired two-tailed t test (DMSO n = 7; 10 n = 6; 50 n = 7; 100 μM n = 8). k Live imaging of the ectopic valve-forming LECs labeled with gata2a:EGFP in embryos treated with selumetinib from 3 to 4 dpf. gata2a:EGFP fluorescence intensities at positions 1–6 are shown. Ectopic gata2a:EGFP expression was found at position 5 in 50 μM treated embryos and positions 5 and 3 in 100 μM treated embryos. All images are anterior to the left, dorsal upward

Article Snippet: Mouse Erk 1/2 antibody (1/2000; Santa Cruz, 514302), rabbit p-Erk1/2 antibody (1/5000; CST, 4370), and rabbit Actb antibody (1/100000; Abclonal, AC026) were used for primary incubation.

Techniques: Inhibition, Immunostaining, Expressing, Two Tailed Test, Imaging, Labeling, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Temporospatial inhibition of Erk signaling is required for lymphatic valve formation

doi: 10.1038/s41392-023-01571-9

Figure Lengend Snippet: efnb2 - ephb4 regulate valve specification through inhibiting Erk signaling. a p-Erk1/2 immunostaining reveals increased Erk signal in FCLV LECs in efnb2a −/− ;efnb2b −/− and ephb4b −/− mutant embryos at 77 hpf. Solid lines, FCLV; yellow dashed lines, other tissues with p-Erk1/2 signals. Arrows indicate the obvious p-Erk1/2 signals in FCLV. Right, enlargement of the boxed regions in the left panels. Scale bars, 20 μm (left) or 10 μm (right). b Relative p-Erk1/2 intensity compared to lyve1b:DsRed2 expression in FCLV in wild-type and mutant embryos. The same batch of experiments with Fig. . Unpaired two-tailed t test (at least 3 independent experiments; wild-type n = 7; efnb2a −/− ;efnb2b −/− n = 7; ephb4b −/− n = 6;). c The changes of Erk activity in the FCLV and aLFL LECs of siblings (Sib) and efnb2a −/− ;efnb2b −/− mutants (Mt) during the valve-forming cell formation. Siblings, n = 7; efnb2a −/− ;efnb2b −/− , n = 7 for the FCLV and n = 9 for the aLFL. Represented images are shown in Supplementary Fig. . Unpaired two-tailed t test, n = 2 independent experiments, one of them is shown here. d , Rescue strategy using small molecules. Treatments started from 2 dpf in 6-well plates and continued until 4 dpf. e Representative results of the treatment of ephb4b −/− mutant larvae with DMSO or 10 μM selumetinib. Selumetinib treatment can efficiently reduce the number of embryos with pericardial edema. Uncropped images can be found in Supplementary Fig. . Scale bar, 1 mm. f The MEK inhibitor selumetinib can restore the pericardial edema defect in ephb4b −/− mutants at 4 dpf. Paired two-tailed t test (at least 3 independent experiments; n = 7). g Selumetinib treatment from 3 dpf induces more gata2a:EGFP positive cells in both the FCLV and aLFL in efnb2a −/− ;efnb2b −/− mutants. Arrowheads, LVs; arrows, FCLV-PHS LVVs. Scale bars, 20 μm. h EGFP fluorescence intensity analyses in ( g ). FCLV, LV and LFL regions for mean intensity analyses are indicated. Unpaired two-tailed t test. i The dynamic expression of Efnb2a on the plasma membrane of FCLV LECs at 60 hpf, valve-forming cells at 77 hpf, and valve cells at 5 dpf during lymphatic valve development. Solid lines, FCLV; dashed lines, LFL. Scale bars, 50 μm (left) or 10 μm (right)

Article Snippet: Mouse Erk 1/2 antibody (1/2000; Santa Cruz, 514302), rabbit p-Erk1/2 antibody (1/5000; CST, 4370), and rabbit Actb antibody (1/100000; Abclonal, AC026) were used for primary incubation.

Techniques: Immunostaining, Mutagenesis, Expressing, Two Tailed Test, Activity Assay, Fluorescence, Membrane