Journal: Nature Communications

Article Title: IL-6 regulates autophagy and chemotherapy resistance by promoting BECN1 phosphorylation

doi: 10.1038/s41467-021-23923-1

Figure Lengend Snippet: a Western blotting was performed for SW48 and LoVo cells separately treated with IL-6 (20 ng/ml) in the absence or presence of CHZ868 (0.2 μM) for 12 h ( n = 2 independent experiments). b SW48 and LoVo cells stably expressing the GFP-LC3B fusion protein were separately treated with IL-6 (20 ng/ml) in the absence or presence of CHZ868 (0.2 μM) for 12 h. Confocal microscopy images are shown. Scale bars, 20 μm ( n = 3 independent experiments). c SW48 and LoVo cells were separately transfected with the JAK2 or control plasmid. After transfection for 24 h, cells were treated with IL-6 (20 ng/ml) for 12 h. Western blotting was performed to examine the expression of LC3B-II and SQSTM1 ( n = 2 independent experiments). d SW48 and LoVo cells stably expressing the GFP-LC3B fusion protein were separately transfected the JAK2 or control plasmid, and after stimulation with IL-6 (20 ng/ml) for 12 h, confocal microscopy images were obtained to measure the number of GFP puncta. Scale bars, 20 μm ( n = 3 independent experiments). e SW48 and LoVo cells were separately transfected with small interfering RNA targeting JAK2 (SiJAK2) or small interfering RNA targeting a negative control gene (SiNC). After transfection for 24 h and following stimulation with IL-6 (20 ng/ml) for 24 h, western blotting was performed to examine the expression of LC3B-II and SQSTM1 ( n = 2 independent experiments). f SW48 and LoVo cells stably expressing GFP-LC3B fusion protein were separately transfected with SiJAK2 and SiNC. After stimulation with IL-6 (20 ng/ml) for 24 h, confocal microscopy images were obtained to measure the number of GFP puncta. Scale bars, 20 μm ( n = 3 independent experiments). Source data are provided in the Source Data file.

Article Snippet: Inhibitors of STAT3 (Stattic, HY-13818) and JAK2 (CHZ868, Cat# HY-18960) were purchased from MedChemExpress.

Techniques: Western Blot, Stable Transfection, Expressing, Confocal Microscopy, Transfection, Control, Plasmid Preparation, Small Interfering RNA, Negative Control

Journal: Nature Communications

Article Title: IL-6 regulates autophagy and chemotherapy resistance by promoting BECN1 phosphorylation

doi: 10.1038/s41467-021-23923-1

Figure Lengend Snippet: a , b Immunoprecipitation (IP) analyses were performed to examine the interaction between BECN1 and JAK2 ( a ) or JAK2 and BECN1 ( b ) in SW48 cells. c Co-IP was performed to examine the relationship between JAK2 and BECN1 in SW48 cells in the absence or presence of IL-6 (20 ng/ml) for 12 h (upper panels). Western blotting was performed on whole-cell extracts (WCEs) (lower panels). d Immunofluorescent staining (IF) analyses of LoVo cells using anti-BECN1 and anti-JAK2 antibodies. Yellow puncta, double-stained BECN1 and JAK2. Scale bars, 20 μm. e Co-IP was performed to examine the interaction of JAK2 and BECN1 in HEK293T cells cotransfected with Flag-BECN1 and HA-JAK2 (WT)/HA-JAK2 (K882E) (upper panels) for 24 h. Western blotting was performed on WCEs (lower panels). f Co-IP was performed to examine the tyrosine phosphorylation of Flag-BECN1 in HEK293T cells cotransfected with Flag-BECN1 and HA-JAK2 (WT)/HA-JAK2 (K882E) (upper panels) for 24 h. Western blotting was performed on WCEs (lower panels). g Co-IP analyses for HA-BECN1 and p-Tyr in HEK293T cells expressing Flag-JAK2, a vector control (Vector), HA-BECN1 WT, HA-BECN1 Y333F, or HA-BECN1 Y338F. Western blotting was performed on WCEs (lower panels). h Modeled complex structure between the ECD domain (magenta, left) and JH1 domain (right), where the JH1 domain consists of four regions: an N-terminal lobe (gray), a C-terminal lobe (cyan), a C helix (red), and an activation loop (yellow). ATP is colored by element, with carbon atoms in green, oxygen atoms in red, nitrogen atoms in blue, and phosphorus atoms in orange. The residues involved in the hydrophobic interaction are shown in pink sphere representation, with atoms shown as sticks. The hydrogen bond is shown by black dashed lines. On the bottom, from left to right, are the enlarged images for the interface in the catalytic site, the interface between the β sheet with the phosphorylation site of the ECD domain and the N-terminal lobe of the JH1 domain, and the interface between the hydrophobic loop of the ECD domain and the C-terminal lobe of the JH1 domain, respectively. i IP analyses for HA-BECN1 and p-BECN1 (Y333) in HEK293T cells expressing HA-BECN1 WT or HA-BECN1 Y333F. Western blotting was performed on WCEs (lower panels). j Western blotting was performed to examine the expression of BECN1 and p-BECN1 (Y333) in LoVo cells in the absence or presence of IL-6 (20 ng/ml) for 12 h. k SW48 cells were separately transfected with SiJAK2 and SiNC. After transfection for 48 h and following stimulation with IL-6 (20 ng/ml) for 12 h, western blotting was performed. Western blots are representative of two independent experiments. WCE whole-cell extract. Source data are provided in the Source Data file.

Article Snippet: Inhibitors of STAT3 (Stattic, HY-13818) and JAK2 (CHZ868, Cat# HY-18960) were purchased from MedChemExpress.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Staining, Phospho-proteomics, Expressing, Plasmid Preparation, Control, Activation Assay, Transfection

Journal: Nature Communications

Article Title: IL-6 regulates autophagy and chemotherapy resistance by promoting BECN1 phosphorylation

doi: 10.1038/s41467-021-23923-1

Figure Lengend Snippet: a Representative results of immunohistochemical staining for p-JAK2 and p-BECN1 (Y333) in the same tumor tissues from 65 clinical CRC patients. Scale bars, 20 μm. b Statistical analysis of the expression of p-JAK2 and p-BECN1 (Y333) in tumor tissues. *** P < 0.001. The values are presented as the means ± s.d.; the p value ( χ 2 -Test) is shown. c Representative results of immunohistochemical staining for p-BECN1 (Y333) in tumor tissues and adjacent normal tissues. Scale bars, 200 μm (top) and 50 μm (bottom). d Statistical analysis of p-BECN1 (Y333) levels in tumor tissues and adjacent normal tissues. *** P < 0.001. Data are presented as the means ± s.d., and the p value was determined by unpaired Student’s t test. e Overall survival was compared between CRC patients with low and high levels of p-BECN1 (Y333) ( n = 44 low p-BECN1 (Y333) levels; n = 57 high p-BECN1 (Y333) levels). Survival data were analyzed by the Kaplan–Meier method and log-rank test. f , g Univariate analysis ( f ) and multivariate analysis ( g ) were performed in Cohort 2 . The bars correspond to 95% confidence intervals. h , i Synergy analysis of the JAK2 inhibitor CHZ868 and chemotherapy drugs (5-Fu or OXA) in LoVo cells. Cells were treated with the indicated concentrations of CHZ868 and 5-Fu ( h ) or OXA ( i ) for 36 h. The combination index (CI) value was examined. The CI value indicates the following: >1.15, synergism = 0.85–1.15, additive effect; and <0.85, antagonism. j Proposed schematic diagram of IL-6-mediated autophagy activation to promote chemotherapy resistance in colorectal cancer. Source data are provided in the Source Data file.

Article Snippet: Inhibitors of STAT3 (Stattic, HY-13818) and JAK2 (CHZ868, Cat# HY-18960) were purchased from MedChemExpress.

Techniques: Immunohistochemical staining, Staining, Expressing, Activation Assay