Journal: iScience

Article Title: Exploring the role of SWI/SNF complex subunit BAF60c in lipid metabolism and inflammation in fish

doi: 10.1016/j.isci.2023.108207

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal Anti-p-ERK1/2 , Cell Signaling Technology , CAT#4370; RIDD: AB_2315112.

Techniques: Virus, Recombinant, Transfection, Sequencing, Plasmid Preparation, Software

Journal: Breast Cancer Research : BCR

Article Title: Long non-coding RNA MIDEAS-AS1 inhibits growth and metastasis of triple-negative breast cancer via transcriptionally activating NCALD

doi: 10.1186/s13058-023-01709-1

Figure Lengend Snippet: The overexpression of NCALD inhibits TNBC cell proliferation and migration in vitro. A-B The efficiency of overexpression and knockdown of NCALD were confirmed by qRT-PCR ( A ) and Western blot ( B ) in MDA-MB-231 and MDA-MB-468 cells. C–E The effects of NCALD overexpression and knockdown on the proliferation of MDA-MB-231 and MDA-MB-468 cells were examined by MTT assay ( C ) and colony formation assays ( D-E ). F Flow cytometry was performed to measure the effect of NCALD overexpression and knockdown on apoptosis. G Transwell and invasion were used to evaluate the motility of MDA-MB-231 and MDA-MB-468 cells transfected with NCALD-overexpressing vector or control vector ( G ) and si-NC or si-MIDEAS-AS1 ( H ). I Western blot analysis of p-ERK1/2, p-NF-κB and TNF-β protein levels in MDA-MB-231 and MDA-MB-468 cells transfected with NCALD overexpression plasmid. J Western blot analysis of p-ERK1/2, p-NF-κB and TNF-β protein levels in MDA-MB-231 and MDA-MB-468 cells transfected with transfected with si-NC or si-NCALD. Data were shown as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The membrane was blocked with 5% nonfat milk at room temperature and incubated overnight with specific primary antibodies at 4 °C: rabbit anti-Fibronectin (Proteintech, Wuhan, China), rabbit anti-N-cadherin (Proteintech, Wuhan, China), rabbit E-cadherin (Proteintech, Wuhan, China), rabbit anti-Vimentin (Proteintech, Wuhan, China), rabbit anti-MMP-9 (Cell Signaling Technology, MA, USA), mouse anti-GAPDH (Servicebio, Wuhan, China), rabbit anti-MATR3 (Proteintech, Wuhan, China), mouse anti-Actin (Servicebio, Wuhan, China), rabbit anti-NCALD (Proteintech, Wuhan, China), rabbit anti-TGF-β (Proteintech, Wuhan, China), rabbit anti-p-p65 (Affinity Biosciences, OH, USA), mouse anti-p65 (Proteintech, Wuhan, China), rabbit anti-p-Erk1/2 (Cell Signaling Technology, MA, USA), rabbit anti-Erk1/2 (Proteintech, Wuhan, China), washed with Tween-20/TBS and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h, followed by enhanced chemiluminescence detection (Vazyme, Nanjing, China), and protein bands were visualized using a ECL detection system (Tanon, Shanghai, China), band density was determined using the ImageJ analyzer software (version 1.48).

Techniques: Over Expression, Migration, In Vitro, Quantitative RT-PCR, Western Blot, MTT Assay, Flow Cytometry, Transfection, Plasmid Preparation

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