Journal: Journal of Functional Foods

Article Title: Hydroxytyrosol promotes random skin flap survival by activating SIRT1-mediated enhancement of autophagy

doi: 10.1016/j.jff.2024.106443

Figure Lengend Snippet: Fig. 5. HT promotes autophagy in skin flaps. (a) Anti-LC3II (red) immunofluorescent antibody was used to stain skin flap tissue slices (scale bar, 20 μm). (b) Calculating the proportion of dermis layer LC3II-positive cells. (c) Random skin flaps stained with CTSD protein using immunohistochemistry (200 × magnification; scale bar, 25 μm). (d) CTSD protein integral absorbance quantification. (e) Beclin1, VPS34, CTSD, LC3II, and P62 expression was found using Western blot analysis. (f) Quantification of Beclin1, VPS34, CTSD, LC3II, and P62 expression levels in every group. Data are shown as mean ± SEM, n = 6, *p < 0.05, **p < 0.01 vs. control group.

Article Snippet: The following businesses were the source of the primary antibodies: MMP9 (10375-2), VEGF (19003-1), HO1 (10701-1), SOD1 (10269-1), anti- SIRT1 (13161-1), VPS34 (12452-1), Caspase-3 (CAPS-3, 19677-1), and cathepsin D (CTSD, 21327-1) were all acquired from Proteintech (Chicago, IL, USA); Cytochrome C (CYC, 14796), anti-mTOR (2983), antiBcl-2 (15071S), anti-p-mTOR (5536S), anti-AMPK (5832S), Bax (32027), and anti-p-AMPK (2535S) were obtained from Cell Signaling Technology (Beverly, USA); SQSTM1/p62 (ab56416) was purchased from Abcam (Cambridge, UK); LC3B (L7543) was sourced from SigmaAldrich Chemical Company (Milwaukee, USA); Cadherin5 (A02632-2) was acquired from Bioss Biotechnology Co., Ltd. (Wuhan, China); GAPDH (AP0063) was obtained from Bioss Biotechnology (Shanghai, China).

Techniques: Staining, Immunohistochemistry, Expressing, Western Blot, Control

Journal: Journal of Functional Foods

Article Title: Hydroxytyrosol promotes random skin flap survival by activating SIRT1-mediated enhancement of autophagy

doi: 10.1016/j.jff.2024.106443

Figure Lengend Snippet: Fig. 7. 3-MA reverses the beneficial effects of HT. (a) Tissue sections of skin flaps were stained using an immunofluorescent antibody called anti-LC3II (red). The scale bar represents a length of 20 μm. (b) Measurement of the proportion of cells positive for LC3II in the dermal layer. The expression of LC3II, Beclin1, VPS34, CTSD, and P62 was detected using Western blot analysis in the control, HT, and HT+3-MA groups. (d) Measurement of the levels of LC3II, Beclin1, VPS34, CTSD, and P62 expression in each group. (e) The Western blot results show the expression of MMP9, VEGF, and Cadherin 5 in each group. (f) Measurement of MMP9, VEGF, and Cadherin 5 expression levels. (g) Western blot analysis was used to assess each group’s SOD1, HO1, and eNOS expression levels. (h) Evaluation of each group’s levels of SOD1, HO1, and eNOS expression. (i) To ascertain the expression levels of CASP3, BAX, and CYC in each group, Western blot analysis was performed. (j) Determining how much CYC, BAX, and CASP3 are expressed in each group. Data are presented as mean ± SEM, n = 6, *p < 0.05, **p < 0.01 compared to the control group. #p < 0:05 and ##p < 0:01 compared to the HT group.

Article Snippet: The following businesses were the source of the primary antibodies: MMP9 (10375-2), VEGF (19003-1), HO1 (10701-1), SOD1 (10269-1), anti- SIRT1 (13161-1), VPS34 (12452-1), Caspase-3 (CAPS-3, 19677-1), and cathepsin D (CTSD, 21327-1) were all acquired from Proteintech (Chicago, IL, USA); Cytochrome C (CYC, 14796), anti-mTOR (2983), antiBcl-2 (15071S), anti-p-mTOR (5536S), anti-AMPK (5832S), Bax (32027), and anti-p-AMPK (2535S) were obtained from Cell Signaling Technology (Beverly, USA); SQSTM1/p62 (ab56416) was purchased from Abcam (Cambridge, UK); LC3B (L7543) was sourced from SigmaAldrich Chemical Company (Milwaukee, USA); Cadherin5 (A02632-2) was acquired from Bioss Biotechnology Co., Ltd. (Wuhan, China); GAPDH (AP0063) was obtained from Bioss Biotechnology (Shanghai, China).

Techniques: Staining, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Deubiquitinase USP15 restricts autophagy and macrophage immunity to Mycobacterium tuberculosis

doi: 10.1101/2025.08.06.668987

Figure Lengend Snippet: A) Western blot of LC3-I and LC3-II and ACTIN in WT and Usp15 KO BV2 cells infected with Mtb. B) Quantification of LC3-II normalized to ACTIN. C) Representative immunofluorescence images of mCherry Mtb (grey or red), LC3 (grey or green) in WT (upper panel) or Usp15 KO (lower panel) BV2 cells. Scale bar is 5 µM. D) Quantification of LC3 co-localization with mCherry Mtb in BV2 cells. E) Schematic of PIK-III inhibition of upstream autophagy initiation via the PI3KC3 complex. F) CFU of WT Mtb in WT or Usp15 KO cells with or without 5 µM of PIK-III normalized for each day to the count on Day 0. For statistical analysis, we used two-way ANOVA and Tukey’s multiple comparison test.* p<0.05, ** p<0.01, *** p<0.001, **** p<0.000.1. Data shown are representative of at least 3 independent experimental replicates.

Article Snippet: PIK-III (MCE, HY-12794) was added to inhibit autophagy immediately post-infection.

Techniques: Western Blot, Infection, Immunofluorescence, Inhibition, Comparison

Journal: Cell reports

Article Title: Subtractive CRISPR screen identifies the ATG16L1/vacuolar ATPase axis as required for non-canonical LC3 lipidation.

doi: 10.1016/j.celrep.2021.109899

Figure Lengend Snippet: Figure 2. v-ATPase is required for M2-induced LC3 lipidation (A) Immunoprecipitation (IP) analysis of endogenous ATG16L1 in HCT116 cells infected for 16 h with PR8. Control 1, lysate from PR8-infected cells incubated with beads to control for unspecific binding; control 2, beads and ATG16L1 antibody. (B) The M2-induced interaction between ATG16L1 and the v-ATPase depends on K490 of the ATG16L1 CTD. HCT116 ATG16L1/ cells reconstituted with WT or K490A mutant FLAG-muATG16L1 were infected with PR8 for 16 h followed by IP with anti-FLAG antibody. (C) Induction of ATG16L1-v-ATPase interaction by M2 depends on M2 ion channel activity. HCT116 ATG16L1/ FLAG-muATG16L1 reconstituted cells were infected with MUd for 16 h. Amantadine was added at 3 days p.i. IP as in (B). (D) Tet-ON M2 cells stably expressing mCherry or mCherry-SopF following treatment with dox or mock treated. M2 expression was detected using the M2- specific antibody 14C2. Black arrow indicates mCherry-SopF-expressing cell; white arrow indicates mCherry-SopF-negative cell. (E) LC3B lipidation analysis in Tet-ON M2 cells stably expressing either mCherry or mCherry-SopF after treatment with either Torin 1 (250 nM for 3 h), dox, or VPS34 IN-1 pretreatment (1 mM for 30 min) followed by monensin (100 mM for 1 h). (F) HCT116 stably expressing mCherry or mCherry-SopF treated as in (E) or infected with PR8 for 16 h (MOI of 10 PFU per cell). (G) Quantification of (E) (right panel) and (F) (left panel). The graph shows fold change in the LC3II/LC3I ratio relative to mCherry DMSO. Bars show mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-LC3B Novus Cat#NB100-2220, RRID:AB_10003146 anti-LC3B Novus Cat#NBP2-46892, RRID:AB_2891060 Mouse anti-Influenza A Virus M2 Protein antibody [14C2] Abcam Cat#ab5416, RRID:AB_304873 Rabbit anti-Influenza A Virus M2 Protein antibody GeneTex Cat#GTX125951, RRID:AB_11170983 Mouse anti-Influenza A Virus Nucleoprotein Abcam Cat#ab20343, RRID:AB_445525 Rabbit anti-Influenza A Virus Nucleoprotein GeneTex Cat#GTX125989, RRID:AB_11168364 anti-GABARP-L1 Proteintech Cat#11010-1-AP, RRID:AB_2294415 anti-GABARP-L2 Abcam Cat#ab126607, RRID:AB_11130165 anti-RalGAPa1 Abcam Cat#ab182570, RRID:AB_2891061 anti-RalGAPb Abcam Cat#ab151139, RRID:AB_2891062 anti-ATG4D Proteintech Cat#16924-1-AP, RRID:AB_2062024 anti-mCherry Novus Cat#NBP1-96752, RRID:AB_11034849 anti-ATG16L1 Cell Signaling Technology Cat#8089, RRID:AB_10950320 anti-ATG16L1 MBL International Cat#PM040, RRID:AB_1278757 anti-ATG12 Santa Cruz Biotechnology Cat#sc271688, RRID:AB_10709301 anti-ATP6V1D Abcam Cat#ab157458, RRID:AB_2732041 anti-ATP6V1A Abcam Cat#ab199326, RRID:AB_2802119 anti-ATP6V1B1/2 Santa Cruz Biotechnology Cat#sc55544, RRID:AB_831844 anti-FLAG Sigma-Aldrich Cat#F1804, RRID:AB_262044 anti-b-Actin Proteintech Cat#20536-1-AP, RRID:AB_10700003 anti-b-Actin Proteintech Cat#66009-1, RRID:AB_2687938 anti-GAPDH Abcam Cat#ab8245, RRID:AB_2107448 anti-tubulin Bio-Rad Cat#MCA77G, RRID:AB_325003 donkey-anti-mouse-IgG-Alexa568 Thermo Cat#A10037, RRID:AB_2534013 goat anti-mouse-IgG-Alexa647 Thermo Cat#A32728, RRID:AB_2633277 IRDye 680RD Goat anti-Mouse IgG LI-COR Biosciences Cat#926-68070, RRID:AB_10956588 IRDye 680RD Goat anti-Rabbit IgG LI-COR Biosciences Cat#926-68071, RRID:AB_10956166 IRDye 800CW Goat anti-Mouse IgG LI-COR Biosciences Cat#926-32210, RRID:AB_621842 IRDye 800CW Goat anti-Rabbit IgG LI-COR Biosciences Cat#926-32211, RRID:AB_621843 IRDye 680RD Goat anti-Rat IgG LI-COR Biosciences Cat#926-68076, RRID:AB_10956590 Chemicals, peptides, and recombinant proteins Torin 1 Selleckchem Cat#S2827 VPS34 inhibitor 1 Selleckchem Cat#S8456 Doxycycline SIGMA Cat#D9891 Monensin SIGMA Cat#M5273 Wortmannin SIGMA Cat#W1628 amantadine hydrochloride SIGMA Cat#A1260 Bafilomycin A1 Abcam Cat#ab120497 Polybrene Santa Cruz Cat#sc134220 G418 Invivogen Cat#ant-gn Blasticidin Invivogen Cat#ant-bl Puromycin Invivogen Cat#ant-pr (Continued on next page) e1 Cell Reports 37, 109899, October 26, 2021

Techniques: Immunoprecipitation, Infection, Control, Incubation, Binding Assay, Mutagenesis, Activity Assay, Stable Transfection, Expressing