il1β  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

doi: 10.1101/2025.04.25.650603

Figure Lengend Snippet: (A) Representative immunofluorescence images of HEK293 cells expressing either an empty vector control or a POMP plasmid for 72h treated with either DMSO or MG132 for 5h. The formation of POMP nuclear puncta is observed only in response to proteasome inhibition and is independent of POMP overexpression. Scale bars = 5 µm. (B) Analysis of the POMP expression levels in the HEK293 cells of the experiment shown in A. While proteasome inhibition leads to a modest but significant increase in POMP levels, POMP overexpression in combination with proteasome inhibition leads to significant and progressively large increase in cellular POMP levels. *p≤0.05, ****p≤0.0001, Welch’s ANOVA test and post-hoc Dunnett’s T3 multiple comparisons test, n=15 (Cntrl DMSO), 14 (Cntrl MG132), 30 (POMP DMSO) and 18 (POMP MG132), mean±SD, FC=fold change. (C) Analysis of the number of nuclear POMP puncta in the HEK293 cells of the experiment shown in A. The formation of POMP nuclear puncta is independent of POMP expression levels in cells and only proteasome inhibition leads to a significant increase in their number. ns=p>0.05, ****p≤0.0001, Welch’s ANOVA test and post-hoc Dunnett’s T3 multiple comparisons test, n=17 (Cntrl DMSO), 16 (Cntrl MG132), 30 (POMP DMSO) and 18 (POMP MG132), mean±SD. (D) Representative immunofluorescence images of HEK293 cells expressing either POMP-Scarlet or NLS-POMP-Scarlet (nuclear localised) overexpression plasmids for 72h treated with either DMSO or MG132 for 5h. Enforcing nuclear localisation of POMP was not sufficient to drive puncta formation, which requires proteasome inhibition.Scale bars = 5 µm. (E) Analysis of the nuclear POMP expression levels in the HEK293 cells of the experiment shown in D. Overexpression of POMP-Scarlet +/- MG132 and NLS-POMP-Scarlet +/- MG132 led to a significant and progressive rise in nuclear POMP levels. ns=p>0.05, *p≤0.05, ***p≤0.001, one-way ANOVA and post-hoc Tukey’s multiple comparisons test, n= 76 (POMP-Scarlet DMSO), 70 (POMP-Scarlet MG132), 56 (NLS-POMP-Scarlet DMSO), 76 (NLS-POMP-Scarlet MG132), mean±SD, FC=fold change. (F) Analysis of the number of nuclear POMP puncta in the HEK293 cells of the experiment shown in D. The formation of POMP nuclear puncta was independent of nuclear POMP levels in cells and only proteasome inhibition led to a significant increase in their number. ns=p>0.05, ****p≤0.0001, one-way ANOVA and post-hoc Tukey’s multiple comparisons test, n=50 (POMP-Scarlet DMSO), 55 (POMP-Scarlet MG132), 54 (NLS-POMP-Scarlet DMSO), 54 (NLS-POMP-Scarlet MG132), mean±SD. (G) Representative immunofluorescence images of hippocampal neurons treated with either DMSO, the proteasome inhibitors MG132, Carfilzomib or the ROS precursor H₂O₂, and stained for MAP2, the nucleolar marker Fibrillarin and the oxidative stress probe CellROX Green. Treatment with H₂O₂ and proteasome inhibition lead to an increase in cellular ROS levels, measured by the increase in CellROX Green fluorescence. Scale bars = 5 µm. (H) Analysis of the CellROX Green fluorescence intensity in the hippocampal neurons of panel G. Treatment with H₂O₂ and proteasome inhibitors led to a significant increase in CellROX Green fluorescence. *p≤0.05, ****p≤0.0001, one-way ANOVA and post-hoc Dunnett’s multiple comparisons test, n=24 (DMSO), 21 (MG132), 27 (Carfilzomib) and 18 (H₂O₂), Boxplots show the median (line), interquartile range (box), and Min-Max whiskers. FC=fold change. (I) Representative immunofluorescence images of hippocampal neurons treated with either DMSO, MG132 alone or MG132 in combination with the reducing agent DTT (1 mM), and immunostained for POMP, MAP2 and the nucleolar marker Fibrillarin. Preventing ROS production with DTT blocked POMP relocalisation to the nucleolus. Scale bar in the low and high-magnification images 10 and 5 µm, respectively. (J) Analysis of neuronal nucleolar POMP levels in the the experiment in I. While proteasome inhibition led to a significant increase in nucleolar POMP levels, blocking ROS production by DTT treatment prevented the nucleolar relocalisation of POMP. ns=p>0.05, ****p≤0.0001, one-way ANOVA and post-hoc Dunnett’s multiple comparisons test, n=54, Boxplots show the median (line), interquartile range (box), and Min-Max whiskers, FC=fold change. (K) Representative non-reducing SDS-PAGE Western blot analysis of cortical neurons treated with either DMSO, MG132 or MG132+1 mM DTT for 5h. Treatment with MG132 led to elevated POMP levels and the appearance of higher molecular species that were redox sensitive and reduced by co-incubation of MG132 with 1 mM DTT on cells. LaminB1 was used as loading control. (L) Analysis of the Western blot shown in K. DTT blocked the relocalization of POMP. The discrepancy in the size of the effects seen for POMP relocalisation I,J and POMP higher molecular weight species levels in K and L can be explained by the re-oxidation POMP during non-reducing SDS-PAGE in atmospheric oxygen. ns=p>0.05, *p≤0.05, RM one-way ANOVA and post-hoc Dunnett’s multiple comparison test, n=4, mean±SD. (M) Analysis of the Western blots shown in of HEK293 treated with pro-inflammatory cytokines (TNFα, INFα, IL1β, GM-CSF) or water control for four days. Treatment with all four cytokines leads to a significant upregulation in P-HSF1 and its downstream targets Hsp70 and POMP. *p≤0.05, **p<0.01, ****p≤0.0001, RM one-way ANOVA and post-hoc Dunnett’s multiple comparison test, n=4, mean±SD. FC=fold change. (N) Analysis of the CellROX Green fluorescence intensity in HEK293 treated with pro-inflammatory cytokines for four days. In all cases treatment leads to a significant increase in CellROX Green fluorescence. ****p≤0.0001, one-way ANOVA and post-hoc Kruskal-Wallis multiple comparisons test, n=57 (Cntrl), 60 (TNFα, IL1β, GM-CSF), 120 (INFα), Boxplots show the median (line), interquartile range (box), and Min-Max whiskers. FC=fold change. (O) Analysis of the nucleolar POMP levels in HEK293 treated with pro-inflammatory cytokines for four days. Treatment with all four cytokines leads to a significant increase in nucleolar POMP levels. *p≤0.05, ****p≤0.0001, one-way ANOVA and post-hoc Dunnett’s multiple comparison test, n=712 (Cntrl), 795 (TNFα), 472 (IFNα), 291 (IL1β), 272 (GM-CSF), mean±SD. FC=fold change.

Article Snippet: The compounds and cytokines used for cell treatment along with vendor and catalog number, the solvent used for reconstitution, the concentration used and treatment duration (in those cases where only one was used) are hereby listed: Carfilzomib (Abcam, ab216469, DMSO, 2 µM), Epoxomicin (Millipore, 324800, DMSO, 2 µM), MG132 (Invitrogen, J63250.MCR, DMSO, 10 µM), Bortezomib (Invitrogen, J60378, DMSO, 1 µM), HSF1B (Axon Medchem, 2101, DMSO, 70 µM, 7 hr), KNK-437 (Sigma-Aldrich, SML0964, DMSO, 100 µM, 5hr in combination with proteasome inhibition), DTT (Millipore, 111474, 1 mM, H2O, 5hr in combination with proteasome inhibition), H2O2 (AlfaAesar, L13235, H2O, 1 mM, 1hr) TNFα (Peprotech, 300-01A-50UG, PBS+0.1%BSA, 0.2 µg/ml, 4 days), IFNα (Abcam, ab48750, PBS+0.1%BSA, 0.2 µg/ml, 4 days), IL1β (CST, 54059, PBS+0.1%BSA, 0.2 µg/ml, 4 days), GM-CSF (CST, 87015, PBS+0.1%BSA, 0.2 µg/ml, 4 days), chloroquine (Fisher Scientific, C230125G, H2O, 100 µM), (Tocris, 0130, DMSO, 10 µM), CNQX (Tocris, 1045, H2O, 20 µM), APV (Tocris, 0106, H2O, 50 µM).

Techniques: Immunofluorescence, Expressing, Plasmid Preparation, Control, Inhibition, Over Expression, Staining, Marker, Fluorescence, Blocking Assay, SDS Page, Western Blot, Incubation, Molecular Weight, Comparison

Journal: bioRxiv

Article Title: The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

doi: 10.1101/2025.04.25.650603

Figure Lengend Snippet: (A) Analysis of experiments like the one shown in . Quantification of nuclear POMP levels in HEK293 cells expressing either an empty vector control (EV) and treated with MG132 (5 hr) or expressing HA-POMP-Myc-Flag (POMP OE) and treated with DMSO (5 hr). POMP overexpression is not sufficient to elevate POMP in the nucleus to the same levels as endogenous POMP following MG132 treatment. *p≤0.05, unpaired two-tailed t-test, n=25 (EV MG132) and 31 (POMP OE DMSO), mean±SD, FC=fold change. (B) Representative images of HEK293 cells expressing HA-Scarlet (Scarlet) or NLS-HA-Scarlet (NLS-Scarlet) treated with DMSO or MG132 for 5 hr. Fusion of an NLS- to Scarlet leads to its strong nuclear accumulation but neither the expression levels or the staining pattern of the construct is affected by proteasome inhibition. Scale bars = 5 µm. (C) Analysis of the nuclear Scarlet levels in the HEK293 cells of experiments like the one shown in B. While NLS-fusion leads to a significant increase in the nuclear levels of Scarlet, MG132 treatment has no effect on the nuclear Scarlet levels for either construct. ns=p>0.05, ****p≤0.0001, one-way ANOVA and post-hoc Tukey’s multiple comparisons test, n=64 (Scarlet DMSO), 60 (NLS-Scarlet DMSO), 63 (Scarlet/NLS-Scarlet MG132) cells, mean±SD, FC=fold change. (D) Analysis of the number of nuclear Scarlet puncta in the HEK293 cells of the experiment shown in B. In general Scarlet does not form nuclear puncta and those few that form due to overexpression are not significantly affected by proteasome inhibition. ns=p>0.05, unpaired two-tailed t-test, n=53 (Scarlet DMSO), 54 (NLS-Scarlet DMSO), 46 (Scarlet MG132), 52 (NLS-Scarlet MG132) cells, mean±SD, FC=fold change. (E) Representative CellROX Green fluorescent images of HEK293 treated with either DMSO, the proteasome inhibitors MG132, Carfilzomib, or 1 mM H₂O₂, as a positive control. Proteasome inhibition and H₂O₂ treatments lead to an increase in CellROX Green fluorescence. Scale bars = 10 µm. (F) Analysis of experiments like the one shown in E. Proteasome inhibition and H₂O₂ treatment lead to a significant increase in CellROX Green fluorescence. *p≤0.05, ***p<0.001, ****p<0.0001, one-way ANOVA with post-hoc Dunnett’s multiple comparisons test, n=16 (DMSO), 17 (Carfilzomib), 20 (MG132, H₂O₂), boxplots show the median (line), interquartile range (box), and Min-Max whiskers. FC=fold change. (G) Multiple sequence alignment of POMP orthologs across species, highlighting conserved cysteine residues. (H) Reducing and non-reducing SDS-PAGE Western blot analysis of primary rat cortical neurons treated with either DMSO, MG132 or Carfilzomib for 5 hr. Blots were probed for proteasome components (PSMA1-7, PSMB5), POMP, ACTB and total protein (loading controls) and transferrin (TF) as a positive control for reduction. Proteasome inhibition leads to the appearance of oligomeric POMP bands, whose intensity decreases following reduction. (I) Line plot of the POMP oligomers intensity profiles under non-reducing conditions. The x-axis reports the molecular weight of the POMP + bands and y-axis their intensity, the dashed line marks the maximal intensity of the peak at ∼25 kDa. Proteasome inhibition leads to the appearance of oligomeric bands. The lines and the areas represent mean and SEM, respectively. n=3 biological replicates. (J) Line plot of the POMP oligomers intensity profiles under reducing conditions. The x-axis reports the molecular weight of the POMP + bands and y-axis their intensity, the dashed line marks the maximal intensity of the peak at ∼25 kDa under non-reducing conditions. The intensity of the peak at ∼25 kDa is decreased by incubation of the extracts with a reducing agent. The lines and the areas represent mean and SEM, respectively. n=3 biological replicates. (K) Analysis of the levels of POMP oligomeric species from experiments shown in H-J. Treatment of the extracts with a reducing agent leads to a significant reduction in the levels of the oligomeric POMP species induced by proteasome inhibition. *p≤0.05, **p<0.01, unpaired two-tailed t-tests, n=3 biological replicates, FC= fold change. (L) Schematic of the POMP oligomerization assay. HA-tagged WT POMP-Scarlet or C36A POMP-Scarlet constructs were co-expressed with a Flag-tagged POMP construct in HEK293 cells that were treated with either DMSO or MG132 for 5 hr. Lysates were subjected to Flag co-IP and the eluates were analysed by non-reducing SDS-PAGE and Western blotting for the HA tag to assay for oligomerization via covalent and non-covalent interactions. (M) Western blot analysis of input and eluate samples from the experiment in L. While WT HA-POMP-Scarlet can interact with POMP-Flag both via non-covalent interactions and disulfide bonds formed via the Cys-residue, the C36A mutant can only interact with the bait via non-covalent interactions and the ability to form higher molecular weight oligomers is entirely lost. Blots of the eluates show that the different constructs express to the same levels and the differences seen after IP cannot be explained by the inputs. (N) Analysis of the POMP levels in the inputs used for the co-IP experiments like the one shown in M. Treatment with MG132 leads to a similar increase in the levels of all three POMP constructs. The C36A mutation does not have any adverse effect on POMP expression. ns=p>0.05, *p≤0.05, one-way ANOVA with post-hoc Šidák’s multiple comparisons tests, n=3 biological replicates, mean±SD. (O) Analysis of HA-POMP-Scarlet levels in the eluates of experiments like the one shown in M. C36A mutation prevents formation of oligomers via disulfide bond formation and leads to a significant reduction in the levels of oligomers formed in response to MG132 treatment. However, MG132 treatment is able to induce a significant increase in the levels of C36A POMP in the eluates via increased non covalent interactions. ns=p>0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with post-hoc Tukey’s multiple comparisons test, n=3 biological replicates, mean±SD. (P) Analysis of the POMP oligomers formed in response to MG132 treatment in the eluates of experiments like the one shown in M. C36A mutation prevents formation of POMP-Scarlet oligomers in the Flag co-IP eluates. **p<0.01, unpaired two-tailed t-test, n=3 biological replicates, mean±SD. (Q) Western blot analysis of HEK293 cells treated with pro-inflammatory cytokines TNFα, INFα, IL1β, GM-CSF for four days. Proteasome activity was probed by in-ge ABP fluorescence. Blots were probed for POMP, HSF1 and its activated form P-HSF1, Hsp70, proteasome subunits (PSMA1-7, PSMB1, PSMB2, PSMB4), immunoproteasome subunits (PSMB8, PSMB9, PSMB10) and GAPDH, as loading control. The numbers reported above the POMP, PSMB4 (pro-form), PSMB9 and PSMB10 represent the log 2 FC average relative to Cntrl for the different treatments. n=4 biological replicates. Quantifications are reported only where at least in one of the treatments log 2 FC average >0.4. For POMP, P-HSF1 and Hsp70 the quantifications are reported in .

Article Snippet: The compounds and cytokines used for cell treatment along with vendor and catalog number, the solvent used for reconstitution, the concentration used and treatment duration (in those cases where only one was used) are hereby listed: Carfilzomib (Abcam, ab216469, DMSO, 2 µM), Epoxomicin (Millipore, 324800, DMSO, 2 µM), MG132 (Invitrogen, J63250.MCR, DMSO, 10 µM), Bortezomib (Invitrogen, J60378, DMSO, 1 µM), HSF1B (Axon Medchem, 2101, DMSO, 70 µM, 7 hr), KNK-437 (Sigma-Aldrich, SML0964, DMSO, 100 µM, 5hr in combination with proteasome inhibition), DTT (Millipore, 111474, 1 mM, H2O, 5hr in combination with proteasome inhibition), H2O2 (AlfaAesar, L13235, H2O, 1 mM, 1hr) TNFα (Peprotech, 300-01A-50UG, PBS+0.1%BSA, 0.2 µg/ml, 4 days), IFNα (Abcam, ab48750, PBS+0.1%BSA, 0.2 µg/ml, 4 days), IL1β (CST, 54059, PBS+0.1%BSA, 0.2 µg/ml, 4 days), GM-CSF (CST, 87015, PBS+0.1%BSA, 0.2 µg/ml, 4 days), chloroquine (Fisher Scientific, C230125G, H2O, 100 µM), (Tocris, 0130, DMSO, 10 µM), CNQX (Tocris, 1045, H2O, 20 µM), APV (Tocris, 0106, H2O, 50 µM).

Techniques: Expressing, Plasmid Preparation, Control, Over Expression, Two Tailed Test, Staining, Construct, Inhibition, Positive Control, Fluorescence, Sequencing, SDS Page, Western Blot, Molecular Weight, Incubation, Co-Immunoprecipitation Assay, Residue, Mutagenesis, Activity Assay

Journal: Cancer Research

Article Title: Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer

doi: 10.1158/0008-5472.CAN-24-1941

Figure Lengend Snippet: Tumor neutrophil ferroptosis was related to a distinctive subset of CD4 T cells enriched in chemoresistant tumors. A, Uniform Manifold Approximation and Projection (UMAP) visualization of tumor-infiltrating CD4 T cells from five chemosensitive and five chemoresistant breast cancers by scRNA-seq. B, Three-dimensional UMAP plot of CD4 T cells colored by chemosensitivity. C, The proportions of different subpopulations of CD4 T cells in sensitive and resistant tumors. D, Heatmap displaying scaled expression of discriminating genes for each cluster of CD4 T cells in scRNA-seq data. E, Heatmap for IL1B expression in CD4 TILs from nine chemosensitive and nine chemoresistant patients by bulk RNA-seq. F, Representative flow cytometry for IL1β and CXCL3 expression in chemosensitive and chemoresistant tumor-infiltrating CD4 T cells. G, Cell death ratio of peripheral neutrophils pretreated with the inhibitors for apoptosis, necrosis, or ferroptosis and cocultured with IL1β + CXCL3 + -d or IL1β + CXCL3 + CD4 T cells. H, Representative flow cytometric images for cellular lipid peroxidation of peripheral neutrophils cocultured with different CD4 T cells. I, Proliferation of tumor-specific CTLs upon exposure to peripheral neutrophils pretreated with IL1β + CXCL3 + or IL1β + CXCL3 + -d CD4 T cells. J, Representative immunofluorescence images (left) and quantification (right) for IL1β and CXCL3 expression in CD4 T cells in chemosensitive and chemoresistant breast cancer sections. Arrows, IL1β + CXCL3 + CD4 T cells. Scale bar, 50 μm. K, Correlation between IL1β + CXCL3 + CD4 T cells and ferroptotic neutrophils in breast tumor specimens. Results are represented as mean ± SD of n = 5 ( A–D and G–I ), n = 9 ( E and F ), or n = 468 ( J ) different patients; for K , n = 468 different patients; ***, P < 0.001 by Student t test ( F ), two-sided one-way ANOVA with the Tukey test ( G , I , and J ) or two-tailed Pearson correlation coefficient test ( K ). Quantification is shown in Supplementary Figs. S4 and S5 ( F and H ).

Article Snippet: Following this, the cells were incubated for 30 minutes at 4°C with specific fluorescent-linked antibodies against human CD3 (BioLegend, cat. #317314, RRID: AB_571909; cat. #317321, RRID: AB_11126166), human CD4 (BioLegend, cat. #317416, RRID: AB_571945; cat. #300512, RRID: AB_314080), human IL1β (eBioscience, cat. #12-7018-82, RRID: AB_466147), human CXCL3 (Abnova, #H00002921-W01P), human CD14 (BioLegend, cat. #301807, RRID: AB_314189), human CD8 (BioLegend, cat. #301005, RRID: AB_314123; cat. #344724, RRID: AB_2562790), human/mouse CD11b (BioLegend, cat. #101227, RRID: AB_893233), human CD66b (BioLegend, cat. #305111, RRID: AB_2563293), human CD71 (BioLegend, cat. #334105, RRID: AB_2271603), human perforin (eBioscience, cat. #48-9994-42, RRID: AB_2574145), human/mouse granzyme B (BioLegend, cat. #372204, RRID: AB_2687028), mouse CD3 (eBioscience, cat. #48-0031-82, RRID: AB_10735092), mouse CD4 (eBioscience, cat. #25-0042-81, RRID: AB_469577), mouse CD8 (eBioscience, cat. #11-0081-82, RRID: AB_464915; cat. #56-0081-80, RRID: AB_494006), mouse perforin (BioLegend, cat. #154405, RRID: AB_2721640), mouse CD14 (BioLegend, cat. #123331, RRID: AB_2734179), mouse Ly-6G (BioLegend, cat. #127613, RRID: AB_1877163), mouse Ly-6C (BioLegend, cat. #128017, RRID: AB_1732093), mouse CD71 (eBioscience, cat. #12-0711-81, RRID: AB_465739), human/mouse Ki67 (BioLegend, cat. #151210, RRID: AB_2716008), cleaved caspase-3 (Cell Signaling Technology, cat. #9603, RRID: AB_11179205) or primary antibodies against mouse CXCL3 (Abcam, cat. #ab220431, RRID: AB_2938758), and mouse IL1β (R&D Systems, cat. #AF-401-SP, RRID: AB_416684).

Techniques: Expressing, RNA Sequencing Assay, Flow Cytometry, Immunofluorescence, Two Tailed Test

Journal: Cancer Research

Article Title: Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer

doi: 10.1158/0008-5472.CAN-24-1941

Figure Lengend Snippet: Ferroptosis-inducing CD4 T cells predisposed tumor neutrophils to ferroptosis through IL1β/IL1R1/NF-κB signaling. A and B, Cell death ratio ( A ) and lipid peroxidation ( B ) of peripheral neutrophils in the noncontact system with Fer-CD4 T cells in the presence of distinct neutralizing antibodies. C, Representative immunofluorescent images for IL1R1 expression in neutrophils with or without different CD4 T-cell encounter. Scale bar, 3 μm. D, Cell death ratio of progenitor-derived neutrophils with or without IL1R1 silencing cocultured with Fer-CD4 T cells. E, Representative fluorescent images for oxidized and nonoxidized lipids of IL1R1 KD or vector-treated progenitor-derived neutrophils cocultured with Fer-CD4 T cells. Scale bar, 5 μm. F, Immunoblot for total and phosphorylated p65 in peripheral neutrophils cocultured with different CD4 T cells. G, Cell death ratio of peripheral neutrophils pretreated with inhibitors for p65, p38, or AKT signaling and cocultured with Fer-CD4 T cells. H, Representative immunoblot for total and phosphorylated p65, p38, and AKT of peripheral neutrophils cocultured with Fer-CD4 T cells with IL1β neutralizing antibody or IL1R1 antagonist (Anak) treatment. I and J, Representative fluorescent images for p65 distribution ( I ) and immunoblot for MBOAT1 ( J ) of peripheral neutrophils cocultured with Fer-CD4 T cells with IL1β neutralization or IL1R1 blockade. Anak, Anakinra. Scale bar, 5 μm. K, Kaplan–Meier survival curves for overall and disease-free survival of patients with breast cancer with low or high proportion of Fer-CD4 T cells. Results are represented as mean ± SD of n = 5 ( A , B , D , and G ) different patients; for K , n = 468 different patients. ***, P < 0.001 by two-sided one-way ANOVA with the Tukey test ( A , D , and G ) or two-sided log-rank test ( K ). For C , E , F , and H–J , independent experiments were performed on three different patients; for I , independent experiments were performed on five different patients, and quantification is shown in Supplementary Fig. S6.

Article Snippet: Following this, the cells were incubated for 30 minutes at 4°C with specific fluorescent-linked antibodies against human CD3 (BioLegend, cat. #317314, RRID: AB_571909; cat. #317321, RRID: AB_11126166), human CD4 (BioLegend, cat. #317416, RRID: AB_571945; cat. #300512, RRID: AB_314080), human IL1β (eBioscience, cat. #12-7018-82, RRID: AB_466147), human CXCL3 (Abnova, #H00002921-W01P), human CD14 (BioLegend, cat. #301807, RRID: AB_314189), human CD8 (BioLegend, cat. #301005, RRID: AB_314123; cat. #344724, RRID: AB_2562790), human/mouse CD11b (BioLegend, cat. #101227, RRID: AB_893233), human CD66b (BioLegend, cat. #305111, RRID: AB_2563293), human CD71 (BioLegend, cat. #334105, RRID: AB_2271603), human perforin (eBioscience, cat. #48-9994-42, RRID: AB_2574145), human/mouse granzyme B (BioLegend, cat. #372204, RRID: AB_2687028), mouse CD3 (eBioscience, cat. #48-0031-82, RRID: AB_10735092), mouse CD4 (eBioscience, cat. #25-0042-81, RRID: AB_469577), mouse CD8 (eBioscience, cat. #11-0081-82, RRID: AB_464915; cat. #56-0081-80, RRID: AB_494006), mouse perforin (BioLegend, cat. #154405, RRID: AB_2721640), mouse CD14 (BioLegend, cat. #123331, RRID: AB_2734179), mouse Ly-6G (BioLegend, cat. #127613, RRID: AB_1877163), mouse Ly-6C (BioLegend, cat. #128017, RRID: AB_1732093), mouse CD71 (eBioscience, cat. #12-0711-81, RRID: AB_465739), human/mouse Ki67 (BioLegend, cat. #151210, RRID: AB_2716008), cleaved caspase-3 (Cell Signaling Technology, cat. #9603, RRID: AB_11179205) or primary antibodies against mouse CXCL3 (Abcam, cat. #ab220431, RRID: AB_2938758), and mouse IL1β (R&D Systems, cat. #AF-401-SP, RRID: AB_416684).

Techniques: Expressing, Derivative Assay, Plasmid Preparation, Western Blot, Neutralization

Journal: Cancer Research

Article Title: Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer

doi: 10.1158/0008-5472.CAN-24-1941

Figure Lengend Snippet: Cross-talk between neutrophils and Fer-CD4 T cells maintained extensive neutrophil ferroptosis. A, Gene Ontology (GO) terms associated with upregulated genes of C2_IL1B cluster in scRNA-seq. B, Gene set enrichment analysis of bulk RNA-seq revealed enrichment of neutrophil chemotaxis genes in chemoresistant CD4 TILs. C, Scheme of chemotaxis assays with Boyden transwell chambers. D, Representative images (left) and quantification (right) of chemotaxis assays for peripheral neutrophils toward CM of different CD4 TILs. Scale bar, 100 μm. E, Migration tracks of neutrophils in μ-slide chemotaxis experiments toward CM of non–Fer-CD4 or Fer-CD4 T cells. F, Representative fluorescent images of cytoskeleton staining (left) and quantification (right) of filopodium-like protrusions (FLP) of peripheral neutrophils in the presence of CM from non–Fer-CD4 or Fer-CD4 T cells. Scale bar, 5 μm. G, Ex vivo tumor slice migration assays for the recruitment of CFSE-labeled neutrophils into breast tumor slices with high or low density of Fer-CD4 T cells. Scale bar, 100 μm. H, Dot plot for the expression of various chemokines in C2_IL1B cells in scRNA-seq. I, ELISA for CXCL3, IL8, and S100A9 release of non–Fer-CD4 or Fer-CD4 T cells. J and K, Migration ( J ) and filopodium-like protrusions ( K ) of peripheral neutrophils in the presence of CM from Fer-CD4 T cells and different antibodies. L, Cell death of peripheral neutrophils in the presence of CM of Fer-CD4 T cells and different antibodies categorized by location (top or bottom chamber of transwell system). M, CXCL3 and IL1β protein level of naïve CD4 T cells by immunoblotting after coculturing with control or ferroptotic neutrophils, which were generated by engagement with non–Fer-CD4 or Fer-CD4 T cells, respectively. Results are represented as mean ± SD of n = 7 ( D ) or n = 5 ( F and I–M ) or n = 6 ( G ). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-sided one-way ANOVA with the Tukey test ( D , F , and J–L ) or Student t test ( G , I , and M ). Representative images are shown in Supplementary Fig. S7 ( J and K ).

Article Snippet: Following this, the cells were incubated for 30 minutes at 4°C with specific fluorescent-linked antibodies against human CD3 (BioLegend, cat. #317314, RRID: AB_571909; cat. #317321, RRID: AB_11126166), human CD4 (BioLegend, cat. #317416, RRID: AB_571945; cat. #300512, RRID: AB_314080), human IL1β (eBioscience, cat. #12-7018-82, RRID: AB_466147), human CXCL3 (Abnova, #H00002921-W01P), human CD14 (BioLegend, cat. #301807, RRID: AB_314189), human CD8 (BioLegend, cat. #301005, RRID: AB_314123; cat. #344724, RRID: AB_2562790), human/mouse CD11b (BioLegend, cat. #101227, RRID: AB_893233), human CD66b (BioLegend, cat. #305111, RRID: AB_2563293), human CD71 (BioLegend, cat. #334105, RRID: AB_2271603), human perforin (eBioscience, cat. #48-9994-42, RRID: AB_2574145), human/mouse granzyme B (BioLegend, cat. #372204, RRID: AB_2687028), mouse CD3 (eBioscience, cat. #48-0031-82, RRID: AB_10735092), mouse CD4 (eBioscience, cat. #25-0042-81, RRID: AB_469577), mouse CD8 (eBioscience, cat. #11-0081-82, RRID: AB_464915; cat. #56-0081-80, RRID: AB_494006), mouse perforin (BioLegend, cat. #154405, RRID: AB_2721640), mouse CD14 (BioLegend, cat. #123331, RRID: AB_2734179), mouse Ly-6G (BioLegend, cat. #127613, RRID: AB_1877163), mouse Ly-6C (BioLegend, cat. #128017, RRID: AB_1732093), mouse CD71 (eBioscience, cat. #12-0711-81, RRID: AB_465739), human/mouse Ki67 (BioLegend, cat. #151210, RRID: AB_2716008), cleaved caspase-3 (Cell Signaling Technology, cat. #9603, RRID: AB_11179205) or primary antibodies against mouse CXCL3 (Abcam, cat. #ab220431, RRID: AB_2938758), and mouse IL1β (R&D Systems, cat. #AF-401-SP, RRID: AB_416684).

Techniques: RNA Sequencing Assay, Chemotaxis Assay, Migration, Staining, Ex Vivo, Labeling, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Generated