Journal: Cell death & disease

Article Title: Mechanistic target of rapamycin complex 1 orchestrates the interplay between hepatocytes and Kupffer cells to determine the outcome of immune-mediated hepatitis.

doi: 10.1038/s41419-022-05487-0

Figure Lengend Snippet: Fig. 5 The activation of mTORC1 in KCs enhances complement alternative system. a Expression heatmap of genes of neutrophils chemotaxis or complement activation analyzed by RNA-seq from Tsc1+/+ and Tsc1-/- BMMs (n = 3 each). b Western blotting result was shown the expression of CFB protein in hepatic tissues from mice. c Left, representative co-immunofluorescent staining images for F4/80 with CFB. Scale bar = 50 μm. Right, quantitative determination of F4/80+ and CFB+ cells among groups as indicated, n = 3. d Western blotting assay showing the abundance for TSC1, CFB, and p-S6 in BMMs. e qRT-PCR analysis showing the CFB mRNA abundance in BMMs, n = 3. f Western blotting assay showing the abundance for TSC1, CFB, and p-S6 in KCs. g qRT-PCR analysis showing the CFB mRNA abundance in KCs, n = 3. h Representative immunofluorescent staining images for C3d. Scale bar = 50 μm. i Representative immunostaining images for C5b-9. Scale bar = 50 μm. j Western blotting assay showing the abundance for Rheb and CFB in BMMs. k Western blotting assay showing the abundance for Rheb and CFB in KCs. l Representative co-immunofluorescent staining images for F4/80 with CFB (white arrows). Scale bar = 100 μm. m Quantitative determination of F4/80+ and CFB+ cells among groups as indicated, n = 3. n Representative immunofluorescent staining images for C3d. Scale bar = 100 μm. o Representative immunostaining images for C5b-9 among groups as indicated. Scale bar = 100 μm. *p < 0.05.

Article Snippet: Three μm sections were cut and immunolabelled with primary antibodies specifically binding F4/80 (cat: 14–4801-82, Invitrogen, USA), p-S6 (Ser235/236) (cat: 4858, Cell Signaling Technology, USA), CD11b (cat: 557396, BD Biosciences), CD3 (cat: 555273, BD Biosciences), ly6b (cat: MCA771G, Bio-Rad, California, USA), Ki67(cat: ab15580, Abcam, Cambridge, UK), cleaved Caspase 3 (cat: 9664, Cell Signaling Technology, USA), CFB (cat: NBP1-89985, Novus; cat: ab192577, Abcam, Cambridge, UK), C3d (cat: AF2655, R&D Systems) or TdT-mediated dUTP-biotin nick end labeling (TUNEL, Promega, Madison, WI).

Techniques: Activation Assay, Expressing, Chemotaxis Assay, RNA Sequencing, Western Blot, Staining, Quantitative RT-PCR, Immunostaining

Journal: Cell death & disease

Article Title: Mechanistic target of rapamycin complex 1 orchestrates the interplay between hepatocytes and Kupffer cells to determine the outcome of immune-mediated hepatitis.

doi: 10.1038/s41419-022-05487-0

Figure Lengend Snippet: Fig. 6 Down regulation of CFB in liver protects against Con-A induced liver injury. a Western blotting assay showing CFB expression in mouse livers after shRNA-CFB injection. b Representative co-immunofluorescent staining images for F4/80 with CFB (white arrows). Scale bar = 100 μm. c The strategy for establishing a mouse model of injection of shRNA-CFB and Con-A administration. d The ALT levels in serum of mice exposed to Con-A for 8 h, n = 6. e Representative HE-stained mouse livers. Scale bar = 100 μm. f Liver sections of were immunofluorescent stained with TUNEL. Scale bar = 200 μm. g Left, representative immunofluorescent staining images for ly6b. Scale bar = 100 μm. Right, quantitative determination of ly6b+ cells among groups as indicated, n = 4. h Left, representative immunofluorescent staining images for C3d. Scale bar = 100 μm. Right, quantitative determination of C3d area in a field of vision, n = 4. i Representative immunostaining images for C5b-9. Scale bar = 100 μm. j Left, representative immunofluorescent staining images for F4/80 (white arrows). Scale bar = 100 μm. Right, quantitative determination of F4/80+ cells among groups as indicated, n = 4. *p < 0.05. k Schematic working model on the role of mTORC1 activation in hepatocytes and KCs in the pathogenesis of ALD.

Article Snippet: Three μm sections were cut and immunolabelled with primary antibodies specifically binding F4/80 (cat: 14–4801-82, Invitrogen, USA), p-S6 (Ser235/236) (cat: 4858, Cell Signaling Technology, USA), CD11b (cat: 557396, BD Biosciences), CD3 (cat: 555273, BD Biosciences), ly6b (cat: MCA771G, Bio-Rad, California, USA), Ki67(cat: ab15580, Abcam, Cambridge, UK), cleaved Caspase 3 (cat: 9664, Cell Signaling Technology, USA), CFB (cat: NBP1-89985, Novus; cat: ab192577, Abcam, Cambridge, UK), C3d (cat: AF2655, R&D Systems) or TdT-mediated dUTP-biotin nick end labeling (TUNEL, Promega, Madison, WI).

Techniques: Western Blot, Expressing, shRNA, Injection, Staining, TUNEL Assay, Immunostaining, Activation Assay

Journal: Frontiers in Immunology

Article Title: Alternative Complement Pathway Is Activated and Associated with Galactose-Deficient IgA 1 Antibody in IgA Nephropathy Patients

doi: 10.3389/fimmu.2021.638309

Figure Lengend Snippet: Glomerular complement factor Bb and C3c deposits were detected in IgAN patients. (A, B) Mesangial positivity can be seen in the complement factor Bb and C3c stains on the glomerulus of a patients with IgA nephropathy. (C, D) No intensity can be detected in the complement factor Bb and C3c stains on the glomerulus of a patient with focal segmental sclerosis. (Magnification: 400X).

Article Snippet: For factor Bb staining, tissue sections were incubated with mouse anti-human complement factor Bb antibody (Bio-Rad, UK).

Techniques:

Journal: Frontiers in Immunology

Article Title: Alternative Complement Pathway Is Activated and Associated with Galactose-Deficient IgA 1 Antibody in IgA Nephropathy Patients

doi: 10.3389/fimmu.2021.638309

Figure Lengend Snippet: Decreased activation of alternative and terminal complement pathway and stabilized renal function after immunosuppression. (A) eGFR decreased slightly among patients who did not receive immunosuppression ( P = 0.028). (B) No change in eGFR among patients received immunosuppression. (C) UPCR tended to decrease slightly among patients who did not receive immunosuppression ( P = 0.051). (D) UPCR decreased significantly in patients who received immunosuppression ( P < 0.0001). (E) Plasma Gd-IgA 1 level tended to decrease 1 months after immunosuppression (N = 17, P = 0.0638) and 3~6 months after immunosuppression (N = 20, P = 0.0019). (F) Plasma C5a significantly decreased 1 (N = 17, P < 0.0001) and 3~6 months after immunosuppression (N = 20, P < 0.0001). (G) Plasma factor Ba level significantly decreased 1 (N = 17, P = 0.0008) and 3~6 months after immunosuppression (N = 20, P = 0.0004). UPCR, urinary protein to creatinine ratio; eGFR, estimated glomerular filtration rate.

Article Snippet: For factor Bb staining, tissue sections were incubated with mouse anti-human complement factor Bb antibody (Bio-Rad, UK).

Techniques: Activation Assay, Clinical Proteomics, Filtration