Journal: eLife

Article Title: m6A modifications regulate intestinal immunity and rotavirus infection

doi: 10.7554/elife.73628

Figure Lengend Snippet: Figure 4. Rotavirus suppresses ALKBH5 expression through NSP1 to evade immune defense. (a) WT mice were infected by RV EW at 8 days post birth. Immunoblotting with antibodies target ALKBH5, FTO, METTL14 and METTL3 in ileum tissue from mice infected with RV EW at two dpi or treated with PBS. (b) Quantitative analysis of (a) (mean ± SEM), Statistical significance was determined by Student’s t-test (*p < 0.05). (c–e) Alkbh5ΔIEC mice and littermate controls were infected by RV EW at 8 days post birth. qPCR analysis of indicated genes expression in ileum (c), viral shedding in feces (d), and viral proteins expression in ileum (e), from Alkbh5ΔIEC mice or littermate controls at 4 days post infection (littermate WT n = 6, Alkbh5ΔIEC n = 5, mean ± SEM). Statistical significance was determined by Student’s t-tests between genotypes (*p < 0.05, NS., not significant). (f) Immunoblotting with antibodies target ALKBH5, NSP1, VP6 and GAPDH in HEK293 cells infected by SA11-4F and SA11-NSP1null (MOI = 1) for 24 hr. (g) Graphical abstract

Article Snippet: METTL3 (abcam, ab195352, 1:2000), METTL14 (sigma, HPA038002, 1:2000), ALKBH5 (sigma, HPA007196, 1:2000), FTO (abcam, ab92821), NSP1 and VP6 (gift from Harry B. Greenberg lab), GAPDH (proteintech), TUBULIN (proteintech), beta- ACTIN (proteintech), Phospho- IRF- 7 (Ser437/438) (D6M2I) (CST), Phospho- TBK1/NAK (Ser172) (D52C2) (CST), TBK1/NAK (D1B4) (CST), and IRF- 7 (D8V1J) (CST) antibodies were used in accordance with the manufacturer’s instructions.

Techniques: Expressing, Infection, Western Blot

Journal: bioRxiv

Article Title: Direct activation of endothelial cells by SARS-CoV-2 nucleocapsid protein is blocked by Simvastatin

doi: 10.1101/2021.02.14.431174

Figure Lengend Snippet: (a) HLMECs were incubated with SARS-CoV2 structural proteins (S, N, and E proteins, 1 μg/mL) and five non-structural proteins (NSP1, NSP3, NSP5, NSP7 and NSP8, 1 μg/mL) for 8 hrs. (b) HLMECs were treated with 1 μg/mL of NP or 10 ng/ml of TNFα for different incubation periods as indicated. (c) HLMECs were incubated with indicated concentrations of NP for 8 hrs. 10 ng/ml of TNFα serves as positive control. (d) Different cultured cells including mouse lung vascular endothelial cells (MEC), A549, 293T, HUVEC, HAEC, HCAEC, HDMEC and HLMEC were treated with NP (1 μg/mL) for 8 hrs. The expression of ICAM-1, VCAM-1 and VE-cadherin was detected by western blot. β-actin was served as loading control. (e) HLMECs were treated with PBS, NP (1 µg/ml), TNFα (10 ng/ml) or LPS (1 µg/ml) for 8 hours. The total RNA was isolated and QPCR was performed for measuring the mRNA levels of TNFα, ICAM-1, VCAM-1, MCP-1 and IL-6. (f) HLMECs were treated with PBS, NP (1 µg/ml) or TNFα (10 ng/ml) for 8 hours and co-cultured with Zombie Red-labeled THP-1 cells for 1h. After washing, the adherent cells were imaged and quantitatively analyzed.

Article Snippet: SARS-CoV-2 NSP1 (97-095), NSP5 (10-116), NSP7 (97-096) and NSP8 (97-097) proteins were obtained from Prosci (Poway, CA).

Techniques: Incubation, Positive Control, Cell Culture, Expressing, Western Blot, Control, Isolation, Labeling

Journal: Cellular signalling

Article Title: SH2D4A inhibits esophageal squamous cell carcinoma progression through FAK/PI3K/AKT signaling pathway.

doi: 10.1016/j.cellsig.2023.110997

Figure Lengend Snippet: Fig. 1. SH2D4A was downregulated in ESCC and lower expression of SH2D4A resulted in an adverse clinical outcome. (A) The mRNA expression of SH2D4A was reduced in GSE53625. (B) The mRNA expression of SH2D4A was reduced in 16 pairs of ESCC tissues detected by RT-qPCR. (C) The expression of SH2D4A were downregulated in ESCC tissues. (D) The relative mRNA expression of SH2D4A was reduced in esophageal carcinoma cell lines detected by RT-qPCR (n = 3 per group). (E) The expression of SH2D4A were downregulated in esophageal carcinoma cell lines. (F, G) Kaplan-Meier analysis of DFS and OS in patients diagnosed with ESCC. All data were provided as mean ± SD. * P < 0.05, and *** P < 0.001.

Article Snippet: Primary antibodies against SH2D4A (15957–1-AP, 1:1000) were purchased from Proteintech.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cellular signalling

Article Title: SH2D4A inhibits esophageal squamous cell carcinoma progression through FAK/PI3K/AKT signaling pathway.

doi: 10.1016/j.cellsig.2023.110997

Figure Lengend Snippet: Fig. 2. SH2D4A overexpression was correlated with a reduction of the proliferation and migration in esophageal carcinoma cells. (A, B) The efficiency of SH2D4A overexpression conducted by lentivirus infection in KYSE-150 and TE-10 (n = 3 per group). (C, D, E) CCK-8 assay, colony formation assay and EDU revealed that SH2D4A overexpression reduce the ability of cells to proliferate (n = 3 per group). (F, G) Transwell migration assay and wound healing assay revealed that SH2D4A overexpression reduce the ability of cells to migrate (n = 3 per group). Scale bar = 100 μm. All data were provided as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Primary antibodies against SH2D4A (15957–1-AP, 1:1000) were purchased from Proteintech.

Techniques: Over Expression, Migration, Infection, CCK-8 Assay, Colony Assay, Transwell Migration Assay, Wound Healing Assay

Journal: Cellular signalling

Article Title: SH2D4A inhibits esophageal squamous cell carcinoma progression through FAK/PI3K/AKT signaling pathway.

doi: 10.1016/j.cellsig.2023.110997

Figure Lengend Snippet: Fig. 3. SH2D4 A knockdown promoted the proliferation and migration of esophageal carcinoma cells. (A, B) The efficiency of SH2D4 A knockdown conducted by lentivirus infection in Eca109 and TE-1 (n = 3 per group). (C, D, E) CCK-8 assay, colony formation assay and EDU revealed that SH2D4A knockdown enhance the ability of cells to proliferate (n = 3 per group). (F, G) Transwell migration assay and wound healing assay revealed that SH2D4A knockdown enhance the ability of cells to migrate (n = 3, per group). Scale bar = 100 μm. All data were provided as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Primary antibodies against SH2D4A (15957–1-AP, 1:1000) were purchased from Proteintech.

Techniques: Knockdown, Migration, Infection, CCK-8 Assay, Colony Assay, Transwell Migration Assay, Wound Healing Assay

Journal: Cellular signalling

Article Title: SH2D4A inhibits esophageal squamous cell carcinoma progression through FAK/PI3K/AKT signaling pathway.

doi: 10.1016/j.cellsig.2023.110997

Figure Lengend Snippet: Fig. 4. SH2D4A regulated FAK/PI3K/AKT signaling pathway in esophageal cancer cells. (A) SH2D4A was enriched in focal adhesion assemblies through KEGG pathway analysis. (B) Correlation analysis between SH2D4A and related molecules in FAK/PI3K/AKT signaling pathway signaling pathway. (C, D) Expression of FAK/PI3K/AKT signaling pathway-related proteins were determined in different cell lines by western blot.

Article Snippet: Primary antibodies against SH2D4A (15957–1-AP, 1:1000) were purchased from Proteintech.

Techniques: Expressing, Western Blot

Journal: Cellular signalling

Article Title: SH2D4A inhibits esophageal squamous cell carcinoma progression through FAK/PI3K/AKT signaling pathway.

doi: 10.1016/j.cellsig.2023.110997

Figure Lengend Snippet: Fig. 5. SH2D4A inhibited esophageal carcinoma cells from proliferating and migrating through the FAK/PI3K/AKT signaling pathway. (A, B, C) CCK-8 assay, colony formation assay and EDU indicated that PF562271 reduced cell proliferation caused by SH2D4A knockdown (n = 3 per group). (D, E) Transwell migration assay and wound healing assay indicated that PF562271 reduced cell migration caused by SH2D4A knockdown (n = 3 per group). (F) PF562271 reduced the facilitation of FAK/PI3K/AKT signaling pathway caused by SH2D4A knockdown. Scale bar = 100 μm. All data were provided as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Primary antibodies against SH2D4A (15957–1-AP, 1:1000) were purchased from Proteintech.

Techniques: CCK-8 Assay, Colony Assay, Knockdown, Transwell Migration Assay, Wound Healing Assay, Migration

Journal: Cellular signalling

Article Title: SH2D4A inhibits esophageal squamous cell carcinoma progression through FAK/PI3K/AKT signaling pathway.

doi: 10.1016/j.cellsig.2023.110997

Figure Lengend Snippet: Fig. 6. SH2D4A knockdown promoted ESCC tumorigenesis in vivo. (A) Tumor sizes were bigger in the si-SH2D4A group than in the si-NC group (n = 5 mice). (B) Growth rate was more rapid in the si-SH2D4A group than in the si-NC group (n = 5 mice). (C) Tumors are heavier in the si-SH2D4A group than in the si-NC group (n = 5 mice). (D) Expression of Ki67 and SH2D4A in tumor tissues were measured by immunohistochemistry. All data were provided as mean ± SD. ** P < 0.01.

Article Snippet: Primary antibodies against SH2D4A (15957–1-AP, 1:1000) were purchased from Proteintech.

Techniques: Knockdown, In Vivo, Expressing, Immunohistochemistry

Journal: Science Advances

Article Title: MAIA, Fc receptor–like 3, supersedes JUNO as IZUMO1 receptor during human fertilization

doi: 10.1126/sciadv.abn0047

Figure Lengend Snippet: ( A ) OBOC library structure (c, d -cysteine; X, 19 l -eukaryotic amino acids except l -cysteine). ( B ) Sperm-OBOC assay. ( C ) Sperm-bead binding (inset: human egg). ( D ) Sperm-bead binding (higher magnification). ( E ) Peptide hits and proportions assigned to functions. ( F ) Sperm (five donors) consistently bound to four specific bead aliquots incubated under specific conditions (fig. S1). Bead 16 hit for FcRL3. ( G ) Sperm bound to resynthesized cAMWNEDc peptide–beads during incubation. ( H ) Sperm-bound bead (higher magnification, bottom left corner; representative sperm highlighted by green cross) and “naked” bead (*). ( I ) IZUMO1 on human sperm. ( J ) Acrosomal antigen 18.6 on human sperm (control). ( K ) Antibody inhibition of sperm binding to resynthesized beads ( n = repetitions) . ( L ) Antibody inhibition of sperm fusion to zona-free hamster eggs ( n = animals, each of 10 to 15 oocytes). ( M ) Gamete interaction proteins on sperm (IZUMO1, SPACA6, DCST1, TMEM95, DCST2, and FIMP) and egg (JUNO, FcRL1, and FcRL3) experienced similar gene evolutionary histories, as shown by ERC sequence analysis. Color intensity reflects the strength of ERC value for that pair of genes. NA, not available.

Article Snippet: HEK293T cells were transfected with FcRL3 plasmid (Origene, SC125617), IZUMO 1R (Origene, SC329497), or a combination of both.

Techniques: Binding Assay, Incubation, Inhibition, Sequencing