Journal: The Journal of Cell Biology

Article Title: V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy

doi: 10.1083/jcb.202206040

Figure Lengend Snippet: Low pH triggers MakA-induced membrane damage and non-canonical autophagy—Supplemental immunofluorescence data. (A) Representative confocal images of MEF cells treated with 250 nM Alexa568-MakA for 16 h and co-stained for endogenous IST1, CHMP2A, or ALIX, as indicated. Nuclei were counterstained with DAPI. Scale bars, 10 µm. Alexa568-MakA refers to Alexa568 labeled monomeric MakA. (B) Representative confocal images of HeLa cells treated with 125 nM Alexa568-MakA for 16 h and co-stained for endogenous ALIX and Gal3 or Gal8, as indicated. Nuclei were counterstained with DAPI. Scale bars, 10 µm. Alexa568-MakA refers to Alexa568 labeled monomeric MakA. (C) Representative confocal images of HeLa cells transfected with mCherry or mCherry-SopF and treated with 250 nM FITC-MakA for 16 h. Cells were co-stained for endogenous LC3 (left) or ALIX (right). Scale bars, 10 µm. FITC-MakA refers to FITC labeled monomeric MakA.

Article Snippet: Gal8 antibody (NBP2-75501, rabbit, IF: 1:400) was purchased from Novus Biologicals.

Techniques: Membrane, Immunofluorescence, Staining, Labeling, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

doi: 10.1186/s13046-021-02095-2

Figure Lengend Snippet: circHMGCS1–016 regulates the miR-1236-3p/GAL-8 and CD73 axis in the ICC cells. A . The overlapped differentiated proteins were shown. For differentially expressed proteins (left panel), the GO and KEGG analysis were performed; B . Western blot for CD73 and GAL-8 proteins in modified RBE and QBC939 cells; C . Putative binding site of miR-1236-3p with respect to GAL-8 and CD73 via StarBase v3.0. D . The luciferase activity of pLG3-CD73 or GAL-8 in the 293 T cells co-transfected with miR-1236-3p. Data are representative of 3 independent tests (** p < 0.01, *** p < 0.001, n.s. p > 0.05); E . The levels of CD73 or GAL-8 proteins were determined by western blot in the ICC cells with different miR-1236-3p or circHMGCS1–016 expression; F . The level of GAL-8 in the supernatant from ICC cells with different miR-1236-3p or circHMGCS1–016 expression was determined; Data are representative of 3 independent tests (*** p < 0.001, n.s. p > 0.05); G . The level of sCD73 in the supernatant from ICC cells with different miR-1236-3p or circHMGCS1–016 expression was determined; Data are representative of 3 independent tests(*** p < 0.001); H . The level of adenosine concentration in the supernatant from ICC cells with different miR-1236-3p or circHMGCS1–016 expression was determined; Data are representative of 3 independent test; I and J. A co-culture showed the supernatant from ICC cells overexpressing circHMGCS1–016 inhibited the CD4 + and CD8 + T cell proliferation; Data are representative of 3 independent tests (*** p < 0.001, n.s. p > 0.05); K. Chemokine chips and ELISA were employed to determine the different chemokines in the supernatant between RBE-control, RBE-circHMGCS1–016 and RBE-shmiR-1236-3p groups

Article Snippet: Rabbit polyclonal to human CD73 antibody (1:1000, ab237757, Abcam, USA), Rabbit monoclonal [EPR3610] to human GAL-8 antibody (1:1000, ab92742, Abcam, USA) and CD4 (1:1000, ab203034, Abcam, USA) were used in immunohistochemistry (IHC).

Techniques: Western Blot, Modification, Binding Assay, Luciferase, Activity Assay, Transfection, Expressing, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

doi: 10.1186/s13046-021-02095-2

Figure Lengend Snippet: GAL-8 and CD73 interference restraints the function of circHMGCS1–016 in ICC cells. A . The relative level of CD73 and GAL-8 was measured by qRT-PCR in the ICC cells; Data are representative of 3 independent tests; B . The expression of CD73 and GAL-8 were modified by lentivirus-mediated knockdown in RBE-circHMGCS1–016 cells. Data are representative of 3 independent tests(*** p < 0.001); C . The efficacy of GAL-8/CD73 interference was analyzed by western blot; D . Invasion assay was performed to detect the invasion ability of RBE-circHMGCS1–016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference (Bar = 200 μm). Data are representative of 3 independent tests(*** p < 0.001); E . Colony formation assay was performed to detect the ability of colony formation in RBE-circHMGCS1–016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference. Data are representative of 3 independent tests(*** p < 0.001); F . The level of GAL-8 in the supernatant of RBE-circHMGCS1–016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference; Data are representative of 3 independent tests(*** p < 0.001); G . The level of adenosine concentration in the supernatant of RBE-circHMGCS1_016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference; Data are representative of 3 independent tests(** p < 0.01); H . A co-culture showed the supernatant from RBE-circHMGCS1–016 cells inhibited the CD4 + and CD8 + T cell proliferation compared to RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference (*** p < 0.001); I . Chemokine chips and ELISA were employed to determine the different chemokines in the supernatant between RBE-circHMGCS1–016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference; J . A model for circHMGCS1–016 driven ICC development and established the immune privilege microenvironment

Article Snippet: Rabbit polyclonal to human CD73 antibody (1:1000, ab237757, Abcam, USA), Rabbit monoclonal [EPR3610] to human GAL-8 antibody (1:1000, ab92742, Abcam, USA) and CD4 (1:1000, ab203034, Abcam, USA) were used in immunohistochemistry (IHC).

Techniques: Quantitative RT-PCR, Expressing, Modification, Western Blot, Invasion Assay, Colony Assay, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

doi: 10.1186/s13046-021-02095-2

Figure Lengend Snippet: The relationship among circHMGCS1–016, CD73 and GAL-8 in ICC tissues. A . CD73 and GAL-8 levels in 40 pairs of ICC and matched adjacent non-tumor tissues. Data was shown as log 2 (T/N) ; B . Representative images for CD73 and GAL-8 staining of ICC and matched adjacent nontumor tissues (Bar = 100 μm); C . Representative images for circHMGCS1–016, CD73, GAL-8, CD4 and CD8 staining of ICC tissues (Bar = 200 μm); D . Correlation analysis showed the positive relationship between circHMGCS1–016 and CD73( R 2 = 0.7379, p = 3.45E-18), circHMGCS1–016 and GAL-8 ( R 2 = 0.6747, p = 5.87E-16). But circHMGCS1_016 expression was adversely related to the level of CD8 ( R 2 = 0.1299, p = 6.45E-17) and CD4 ( R 2 = 0.2157, p = 1.17E-14). The relationship between CD8 and CD73( R 2 = 0.3711, p = 1.81E-12), CD8 and GAL-8 ( R 2 = 0. 4244, p = 2.12E-23) was also analyzed (*** p < 0.001)

Article Snippet: Rabbit polyclonal to human CD73 antibody (1:1000, ab237757, Abcam, USA), Rabbit monoclonal [EPR3610] to human GAL-8 antibody (1:1000, ab92742, Abcam, USA) and CD4 (1:1000, ab203034, Abcam, USA) were used in immunohistochemistry (IHC).

Techniques: Staining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

doi: 10.1186/s13046-021-02095-2

Figure Lengend Snippet: Higher levels of circHMGCS1–016 correlated with resistance to anti-PD1 therapy in mice and ICC patients. A . Representative images of the RBE orthotopic ICC tumors from humanized NSG mice ( n = 3 /group). B . Tumor growth volume of the RBE orthotopic planted humanized mice from each group (*** p < 0.001, ** p < 0.01); C . The level of GAL-8 in the serum of the RBE orthotopic ICC tumors from each group; Data are representative of 3 independent tests (*** p < 0.001, * p < 0.05, n.s. p > 0.05); D . The level of adenosine concentration in the serum of mice planted with the RBE orthotopic ICC tumors; Data are representative of 3 independent tests(** p < 0.01, * p < 0.05); E . The CD8 + and CD4 + T cells in the blood of mice planted with the RBE orthotopic ICC tumors (*** p < 0.001); F. At study endpoint, the CD8 + and CD4 + T cells in IgG and PD-1 treatment group were analyzed by immunohistochemistry (Bar = 200 μm); G . Representative ICC cases from 12 patients who received PD-1 antibody treatment were analyzed by IHC staining for CD8 and circHMGCS1–016 (Bar = 200 μm); H . Twelve patients were divided into two groups according to circHMGCS1–016 expression, and patients in CR, PR, SD, and PD were shown in each group. I . The efficacy of PD1 antibody immunotherapy was assessed by MRI based on RECIST1.1. J . The number of CD8 + cells was significantly different between two groups(** p < 0.01)

Article Snippet: Rabbit polyclonal to human CD73 antibody (1:1000, ab237757, Abcam, USA), Rabbit monoclonal [EPR3610] to human GAL-8 antibody (1:1000, ab92742, Abcam, USA) and CD4 (1:1000, ab203034, Abcam, USA) were used in immunohistochemistry (IHC).

Techniques: Concentration Assay, Immunohistochemistry, Expressing