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: Cell Reports

Article Title: Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity

doi: 10.1016/j.celrep.2018.08.006

Figure Lengend Snippet:

Article Snippet: The human/mouse Anti-UCP1 antibody (MAB6158, monoclonal Mouse IgG 2B Clone # 536435, R&D Systems) was conjugated to Alexa647 (ThermoFisher Alexa Fluor 647 antibody labeling kit A20186) and incubated with adipocytes at 1:300 for 1 h. Mouse IgG2B Alexa Fluor 647-conjugated Isotype Control antibody (R&D systems) was used as a control.

Techniques: Control, Recombinant, Blocking Assay, Antibody Labeling, Software, Flow Cytometry

Journal: Diabetologia

Article Title: Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

doi: 10.1007/s00125-019-04974-y

Figure Lengend Snippet: Anti-insulin B cells repopulate pancreatic islets more rapidly than insulin-negative B cells after anti-CD20 treatment. Groups of VH125.hCD20/NOD mice, aged 6–8 weeks old, were injected with 2H7 anti-CD20 or IgG isotype control. Spleen, PLNs and pancreatic islets were analysed for insulin-positive and insulin-negative B cells at 8 weeks and 12 weeks post depletion by flow cytometry. ( a – f ) No. of cells from IgG control-treated (black circles) and 2H7-treated (grey squares) mice for insulin-negative B cells ( a – c ) and insulin-positive B cells ( d – f ) from spleen ( a , d ) PLNs ( b , e ) and islets ( c , f ). ( g – i ) Percentage of B cells repopulated at 8 and 12 weeks after treatment from spleen ( g ), PLNs ( h ) and islets ( i ) of mice shown in ( a – f ). Percentages were calculated as individual numbers from each 2H7-treated mouse / mean number from all IgG control antibody-treated mice. Horizontal lines represent the median value. Data represent three independent experiments. At 8 weeks, n = 7 (spleen), n = 7 (PLNs) and n = 9 (islets) for control IgG-treated mice and n = 10 (spleen), n = 9 (PLNs) and n = 12 (islets) for 2H7-treated mice. At 12 weeks, n = 8 (spleen), n = 6 (PLNs) and n = 7 (islets) for control IgG and n = 11 (spleen), n = 7 (PLNs) and n = 11 (islets) for 2H7-treated mice. * p < 0.05 (one-way ANOVA)

Article Snippet: Female VH125.hCD20/NOD mice, 6–8 weeks of age were chosen at random to receive either anti-hCD20 antibody (clone 2H7; Bio-XCell, West Lebanon, NH, USA) or control IgG2b antibody (clone MPC-11; Bio-XCell [ , , ]), as described previously [ ].

Techniques: Injection, Control, Flow Cytometry

Journal: Diabetologia

Article Title: Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

doi: 10.1007/s00125-019-04974-y

Figure Lengend Snippet: CD138 int anti-insulin B cells are enriched in pancreatic islets after anti-CD20 treatment. Groups of 6- to 8-week-old VH125.hCD20/NOD mice were injected with 2H7 anti-CD20 or IgG isotype control. Groups of mice ( n = 2 or 3 per group) were pooled and insulin + B cells from pancreatic islets were analysed for four different populations based on CD138 expression: CD138 − (blue); CD138 int IgM + (orange); CD138 int IgM lo (grey) and CD138 hi IgM lo (red). ( a , b ) Representative flow plots showing gating on live CD3 − CD11b − CD11c − ( a ) and graph showing the overall percentages of the four different populations ( b ). ( c , d ) Representative flow plots showing insulin − CD19 + , insulin + CD19 + and insulin + CD19 − cells ( c ) and graph showing the overall percentages of these cells ( d ); 2H7 (black circles), IgG (grey circles). ( e ) Representative flow plots showing CD138 and IgM expression in insulin + CD19 + and insulin + CD19 − cells. ( f , g ) Graphs showing CD138 and IgM populations on insulin + CD19 + ( f ) and insulin + CD19 − cells ( g ) ( n = 5 groups for control IgG treatment; n = 4 groups for 2H7 treatment). Horizontal lines represent the median values. Data represent two independent experiments. * p < 0.05 (one-way ANOVA)

Article Snippet: Female VH125.hCD20/NOD mice, 6–8 weeks of age were chosen at random to receive either anti-hCD20 antibody (clone 2H7; Bio-XCell, West Lebanon, NH, USA) or control IgG2b antibody (clone MPC-11; Bio-XCell [ , , ]), as described previously [ ].

Techniques: Injection, Control, Expressing