Journal: Acta Pharmaceutica Sinica. B

Article Title: Identification of anthelmintic parbendazole as a therapeutic molecule for HNSCC through connectivity map-based drug repositioning

doi: 10.1016/j.apsb.2021.12.005

Figure Lengend Snippet: Parbendazole inhibits tubulin polymerization. (A) is the result of immunofluorescence assay which displays the microtubule structure of cells. Red (Alexa Fluor® 555) was used to mark tubulin, and blue (DAPI) was used to mark cell nuclei. Images were obtained by Laser Confocal Microscope (LSM780). The scale bar was 20 μm; (B) shows compounds inhibited tubulin polymerization in vitro . The spontaneous polymerization of tubulin in the absence of the compound was used as a negative control; (C) is the result of Western blotting of the amount of unpolymerizated and polymerizated tubulin; (D) shows unpolymerizated/polymerizated tubulin ratio. Data is represented as mean ± SEM ( n = 3, ∗ P < 0.05).

Article Snippet: This test was performed using the tubulin polymerization kit in accordance with the manufacturer's protocol (BK011P, Cytoskeleton, Inc.).

Techniques: Immunofluorescence, Microscopy, In Vitro, Negative Control, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Anlotinib Benefits the α PDL1 Immunotherapy by Activating ROS/JNK/AP-1 Pathway to Upregulate PDL1 Expression in Colorectal Cancer

doi: 10.1155/2022/8965903

Figure Lengend Snippet: Anlo increased the expression of PDL1 by ROS/JNK/AP-1 pathway in vitro . (a) ROS production was determined and analyzed by flow cytometry. (b) Representative western blots of HSP90, T-JNK, P-JNK, P-C-FOS, P-C-JUN, IFN- α , IFN- β , and IFN- γ . (c) The expression of mRNA levels of IFN- α , IFN- β , and IFN- γ . (d) RNA sequence data analysis of CT26 tumor.

Article Snippet: After blocking with skim milk for 1 hour in TBST, the membranes were incubated with the primary antibodies included anti-PDL1 (#1-76769, Novus, USA, 1 : 1000), anti-PDL1 (#DF6526, affinity, 1 : 1000), anti-HSP90 (#4877, CST, Boston, MA, USA, 1 : 1000), anti-IFN α (#DF6086, affinity, 1 : 1000), anti-IFN β (#ab218229, Abcam, 1 : 1000), Anti-IFN γ (#DF6045, affinity, 1 : 1000), anti-JNK2 (#9258, CST, 1 : 1000), P-JNK (#4668, CST, 1 : 1000), P-c-fos (#5348, CST, 1 : 1000), and P-c-jun (#2361, CST, 1 : 1000) at 4°C overnight.

Techniques: Expressing, In Vitro, Flow Cytometry, Western Blot, Sequencing

Journal: Nature Microbiology

Article Title: Evolution of enhanced innate immune suppression by SARS-CoV-2 Omicron subvariants

doi: 10.1038/s41564-023-01588-4

Figure Lengend Snippet: a – g , Calu-3 infection with 2,000 E copies per cell of Delta (yellow, Ο), BA.1 (blue, Ο), BA.2 (blue, Δ), BA.4 (purple, O) and BA.5 (purple, Δ), n = 3: mean viral E copies at 2 h.p.i. across three independent experiments ( a ); viral replication over time measured by RT–qPCR for intracellular E copies per microgram RNA ( b ); infection levels measured by nucleocapsid expression (% N+ by flow cytometry) ( c ); expression of IFNB , CXCL10 , IFIT1 , IFIT2 , RSAD2 , MX1 , MX2 and DDX58 in infected cells over time ( d ); IFNβ ( e ) and CXCL10 ( f ) secretion from infected Calu-3 cells measured by ELISA at 48 h.p.i.; rescue of viral replication by JAK1-inhibitor ruxolitinib in Calu-3 cells at 48 h.p.i., where relative infection levels are shown across three independent experiments determined by E copies per microgram RNA normalized to the median infection level of the untreated control ( g ). h – k , Primary bronchial HAEs were infected with the indicated variants at 1,500 E copies per cell: viral replication measured by intracellular E copies at 72 h.p.i. ( h ) and viral release into apical washes over time ( i ), with three biological replicates shown; expression of IFNB , CXCL10 , IFIT1 , IFIT2 , DDX58 and RSAD2 in HAEs at 72 h.p.i., with six biological replicates shown ( j ); intracellular viral E copies in HAEs in the presence or absence of 5 μM ruxolitinib at 72 h.p.i., with three biological replicates shown ( k ). For a , one-way analysis of variance (ANOVA) with Bonferroni post-test was used. n.s., not significant at P > 0.05 for all comparisons. For b – h and j , one-way ANOVA and Dunnett’s post-test were used. For i , two-way ANOVA with a Bonferroni post-test was used. For k , one-tailed unpaired Student’s t -test was used. Replicate measurements from one of three independent experiments. Fold change over mock is shown. Mean ± s.e.m. or individual datapoints are shown. h.p.i., hours post infection.

Article Snippet: IFNβ, IFNλ1/IFNλ3 and CXCL10 were measured using Human IFN-β Quantikine ELISA Kit, Human IL-29/IL-28B (IFNλ1/IFNλ3) DuoSet ELISA or Human CXCL10/IP-10 DuoSet ELISA reagents (Bio-Techne R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Quantitative RT-PCR, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, One-tailed Test