Journal: Cell Research

Article Title: Targeting ATAD3A-PINK1-mitophagy axis overcomes chemoimmunotherapy resistance by redirecting PD-L1 to mitochondria

doi: 10.1038/s41422-022-00766-z

Figure Lengend Snippet: a Left, immunostaining of PD-L1 (green) and TOM20-labeled mitochondria (red) in BT549 human TNBC cells with or without ATAD3A-knockdown (shATAD3A#1 and shATAD3A#2). Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 ( n = 5 fields, t -test). b Immunoblot of PD-L1 in the cytoplasm and mitochondria of control and ATAD3A-knockdown BT549 cells. TOM20 and Tubulin were used as mitochondria and cytoplasm protein controls, respectively. Cyto, cytoplasm; mito, mitochondria. c Flow cytometry (left) and quantification (right) of surface PD-L1 in control and ATAD3A-knockdown BT549 cells ( n = 3, one-way ANOVA). d Venn diagram depicting overlapped genes for the interaction protein of ATAD3A set (BioGRID, RP5-832C2.1), the protein localization to mitochondrion set (GOBP 0070585) and the intrinsic component of mitochondrial membrane (GOCC 0098573). e Immunoblot of PINK1 in control and ATAD3A-knockdown BT549 cells. f Immunoblot of PD-L1 and PINK1 in HEK293T cells overexpressing PD-L1 (OE-PD-L1) and control cells (OE-Control), assessed after immunoprecipitation with immunoglobulin G (IgG) or antibody to PINK1. g Protein direct interaction analysis of the intracellular domain of PD-L1 (ICD) and PINK1 in vitro. Purified Flag-labeled full-length PINK1 was incubated with Biotin-labeled PD-L1 ICD domain, followed by streptavidin pull-down and immunoblot. h Schematic diagram of Flag-labeled full-length (FL) and truncated mutants with indicated domains (amino acids 1–155, amino acids 156–320, amino acids 321–509, amino acids 510–581) of PINK1. MTS, mitochondrial targeting sequence; N-lobe, kinase domain N; C-lobe, kinase domain C; CTD, C-terminal domain. i Protein direct interaction analysis of the intracellular domain of PD-L1 (ICD) and truncated PINK1 mutants in vitro. Purified Flag-labeled full-length and truncated PINK1 were incubated with Biotin-labeled PD-L1 ICD domain, followed by streptavidin pull-down and immunoblot. The estimated size of PINK1-4 (amino acids 510–581) which did not express in HEK293T cells was labeled with asterisk. j Immunoblot of PD-L1 in the cytoplasm and mitochondria of MDA-MB-231 cells with or without PINK1-knockdown (shPINK1#1 and shPINK1#2). TOM20 and Tubulin were used as mitochondria and cytoplasm protein controls. Cyto, cytoplasm; mito, mitochondria. k Left, co-localization of PD-L1 (green) and TOM20 (red) in control, ATAD3A knockdown, PINK1 knockdown or ATAD3A and PINK1 double knockdown BT549 cells. Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 ( n = 5 fields, one-way ANOVA). l Immunoblot of PD-L1 in the cytoplasm and mitochondria of control, ATAD3A-knockdown, PINK1-knockdown or ATAD3A and PINK1 double knockdown BT549 cells. m Immunoblot of indicated proteins in PD-L1-transfected HEK293T cells with or without PINK1 overexpression. n Immunoblot of PD-L1 in control and PINK1-knockdown (shPINK1#1 and shPINK1#2) BT549 cells. o Immunoblot of PD-L1 in BT549 cells transfected with control shRNA or shATAD3A (shATAD3A#1 and shATAD3A#2). p Immunoblot of total PD-L1 in control, ATAD3A-knockdown, PINK1-knockdown or ATAD3A and PINK1 double knockdown BT549 cells. q Immunoblot of PD-L1 in control and ATAD3A-knockdown BT549 cells treated with 20 μM CHX for indicated times. h, hours. r Quantification of PD-L1 intensity in immunoblot in control and ATAD3A-knockdown BT549 cells. s Immunoblot of PD-L1 in control and ATAD3A-knockdown MDA-MB-231 cells incubated with 20 nM BafA1 for indicated times. h, hours. Data are representative of at least two independent experiments and are shown as means ± SD. See also Supplementary information, Figs. and .

Article Snippet: Mouse IgG2b (APC, clone MPC-11, BioLegend) and rat IgG2b (APC, clone RTK4530, BioLegend) were used as controls respectively.

Techniques: Immunostaining, Labeling, Knockdown, Western Blot, Control, Flow Cytometry, Membrane, Immunoprecipitation, In Vitro, Purification, Incubation, Sequencing, Transfection, Over Expression, shRNA

Journal: Cell Research

Article Title: Targeting ATAD3A-PINK1-mitophagy axis overcomes chemoimmunotherapy resistance by redirecting PD-L1 to mitochondria

doi: 10.1038/s41422-022-00766-z

Figure Lengend Snippet: a – h BALB/c mice were inoculated orthotopically with 5 × 10 4 4T1 cells transfected with control shRNA (shControl) or shRNA for Atad3a (shAtad3a#1 and shAtad3a#2). a , b The endpoint tumor images ( a ) and volume ( b ) of tumors formed by control and Atad3a-knockdown cells in BALB/c mice ( n = 6, one-way ANOVA). c Left, IHC staining of Atad3a and PD-L1 on serial sections of tumors formed by control and Atad3a-knockdown cells. Scale bars, 50 μm. Right, IHC score of Atad3a in control and Atad3a-knockdown tumors ( n = 6 fields, t -test). d IHC score of PD-L1 in control and Atad3a-knockdown tumors ( n = 6 fields, t -test). e Quantification of the percentage of tumor-infiltrating CD8 + T cells in tumors formed by control and Atad3a-knockdown cells by flow cytometry ( n = 5, t -test). f Quantification of the percentages of tumor-infiltrating IFNγ + CD8 + T cells and IFNγ + CD4 + T cells by flow cytometry ( n = 5, t -test). g Ratio of CD8 + cytotoxic T lymphocytes to CD4 + CD25 + Foxp3 + T reg cells ( n = 5, t -test). h Quantification of the percentages of PD-1 + TIM-3 + CD8 + T cells (left) and PD-1 + TIM-3 + CD4 + T cells (right) by flow cytometry ( n = 5, t -test). i – m BALB/c mice were inoculated orthotopically with 5 × 10 4 4T1 cells transfected with control shRNA (shControl) or shRNA specific for Atad3a (shAtad3a), Pink1 (shPink1) or both (shAtad3a + shPink1). i , Left, the endpoint images of tumors formed by control, Atad3a-knockdown, Pink1-knockdown or Atad3a and Pink1 double knockdown 4T1 cells in BALB/c mice. Right, immunoblot of Atad3a and Pink1 in these 4T1 cells. j The volume of tumors mentioned above ( n = 6, one-way ANOVA). k Quantification of the percentage of tumor-infiltrating CD8 + T cells by flow cytometry ( n = 5, one-way ANOVA). l Quantification of the percentage of tumor-infiltrating IFNγ + CD8 + T cells by flow cytometry ( n = 5, one-way ANOVA). m Quantification of the percentage of PD-1 + TIM-3 + CD8 + T cells by flow cytometry ( n = 5, one-way ANOVA). n – s 4T1 tumors formed by control and Atad3a-knockdown cells were established orthotopically in BALB/c mice and received vehicle, anti-PD-L1 antibody (PD-L1 mAb), paclitaxel (PTX) or combined anti-PD-L1 antibody with paclitaxel treatment (PD-L1 mAb + PTX). IgG2b and saline were used as controls. n Experimental protocol. o , p The endpoint tumor images ( o ) and the volume ( p ) of tumors ( n = 6, one-way ANOVA). q – s Quantification of the percentages of tumor-infiltrating CD8 + T cells ( q ), IFNγ + CD8 + T cells ( r ) and PD-1 + TIM-3 + CD8 + T cells ( s ) in 4T1 tumors formed by control and Atad3a-knockdown cells received treatments as described above, determined by flow cytometry ( n = 5, one-way ANOVA). t Schematic model. Patients with PD-L1-positive TNBC could be divided into two groups based on ATAD3A expression. Patients with ATAD3A-high tumors might respond more poorly to ICIs plus paclitaxel therapy, and inhibition of ATAD3A is required to improve clinical outcome. Patients with ATAD3A-low tumors might benefit significantly from ICIs plus paclitaxel combination therapy. See also Supplementary information, Figs. – .

Article Snippet: Mouse IgG2b (APC, clone MPC-11, BioLegend) and rat IgG2b (APC, clone RTK4530, BioLegend) were used as controls respectively.

Techniques: Transfection, Control, shRNA, Knockdown, Immunohistochemistry, Flow Cytometry, Western Blot, Saline, Expressing, Inhibition

Journal: bioRxiv

Article Title: The binding mechanism of Streptococcus suis accessory virulence factor and adhesin SadP to globotetraosylceramide

doi: 10.1101/2020.05.29.122762

Figure Lengend Snippet: POPC/glycolipid liposomes (4, 0.4 and 0.04 nmol/dot) and POPC/cholesterol/globosylceramide liposomes (4, 0.4 and 0.04 nmol/dot) were applied onto PVDF membrane. WT, His-tagged SadP(125-329) type P N ; N285D, site-specific mutant of SadP(125-329); Replicate membranes containing dotted liposomes with or without cholesterol (see Methods) were probed with SadP and SadP N285D . The bound proteins were detected with anti-His and HRP-labelled antibodies as described in the methods and membrane was imaged with FujiLAS-4000. The globo series glycolipids used were: Gb4, d18:1-24:0 + d18:1 – 16:0; Gb3/1, Gb3 d18:1-24:0; Gb3/2, Gb3 d18:1-16:0; Gb3/3, Gb3 d18:1-h16:0.

Article Snippet: Finally, streptavidin donor beads were added (20 μg/ml) and the mixtures were incubated for 30 mins and were then measured with the Ensight multimode reader (Perkin Elmer) using excitation wavelength of 680 nm with donor beads and measurement of emission wavelength of 615 nm from AlphaLISA anti-HIS acceptor beads.

Techniques: Liposomes, Membrane, Mutagenesis