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( A ) Schematic of CM from chemotherapy-treated cancer cells for subsequent treatment on CAFs in vitro. ( B ) Flow cytometry evaluating CM effects on αSMA high/+ CAFs and αSMA − PDGFRβ + iCAF conversion, in response to (i) base medium (2% FBS DMEM), (ii) CM from untreated T24 (CM-Veh), and (iii) CM from gemcitabine-treated T24 (CM-Gem). ( C and D ) Bar graphs quantifying αSMA high/+ CAFs (C) and αSMA − PDGFβ + iCAFs (D) upon treatment with CM-Veh and CM-Gem. ( E ) Flow cytometry histogram illustrating higher IL-6 expression in αSMA − PDGFRβ + iCAFs (blue) and αSMA + PDGFRβ + hybrid i/my CAFs (red) than other CAFs (green and yellow). ( F to H ) Western blot analysis of Casp1-dependent pyroptosis and apoptosis in gemcitabine-treated T24 cells by immunoblotting (IB): (F) Casp1 full-length (FL) protein, (G) Casp1 p20 and p10 cleavage (Cl) products indicating enzymatic activity, and (H) Caspase-3 (Casp3) FL, Cl-Casp3, and DNA repair protein poly(ADP-ribose) polymerase 1 (PARP1) FL and Cl PARP1 (Cl-PARP1), as apoptosis markers. ( I ) Flow cytometry of <t>DAPI</t> and annexin V costaining in WT and Casp1 knockout (Casp1 KO) T24 cells upon gemcitabine treatment. ( J ) Bar graph quantifying fractions in (I), showing reduced lytic cell death (DAPI + /annexin V − ; red) in Casp1 KO cells ( * P = 0.0265). ( K ) Flow cytometry analyzing CM from gemcitabine-treated WT or Casp1 knockout (Casp1 KO) T24 cells in the conversion between αSMA high/+ CAFs and αSMA − PDGFβ + iCAFs. ( L ) Corresponding IF costaining illustrating PDGFRβ (red) and αSMA (green) in CAFs exposed to CM-Gem from T24 WT and Casp1 KO cells. ( M ) Bar graph quantifying PDGFRβ + iCAFs, showing significant reduction after exposure to CM-Gem from Casp1 KO versus WT cells.
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( A ) Schematic of CM from chemotherapy-treated cancer cells for subsequent treatment on CAFs in vitro. ( B ) Flow cytometry evaluating CM effects on αSMA high/+ CAFs and αSMA − PDGFRβ + iCAF conversion, in response to (i) base medium (2% FBS DMEM), (ii) CM from untreated T24 (CM-Veh), and (iii) CM from gemcitabine-treated T24 (CM-Gem). ( C and D ) Bar graphs quantifying αSMA high/+ CAFs (C) and αSMA − PDGFβ + iCAFs (D) upon treatment with CM-Veh and CM-Gem. ( E ) Flow cytometry histogram illustrating higher IL-6 expression in αSMA − PDGFRβ + iCAFs (blue) and αSMA + PDGFRβ + hybrid i/my CAFs (red) than other CAFs (green and yellow). ( F to H ) Western blot analysis of Casp1-dependent pyroptosis and apoptosis in gemcitabine-treated T24 cells by immunoblotting (IB): (F) Casp1 full-length (FL) protein, (G) Casp1 p20 and p10 cleavage (Cl) products indicating enzymatic activity, and (H) Caspase-3 (Casp3) FL, Cl-Casp3, and DNA repair protein poly(ADP-ribose) polymerase 1 (PARP1) FL and Cl PARP1 (Cl-PARP1), as apoptosis markers. ( I ) Flow cytometry of DAPI and annexin V costaining in WT and Casp1 knockout (Casp1 KO) T24 cells upon gemcitabine treatment. ( J ) Bar graph quantifying fractions in (I), showing reduced lytic cell death (DAPI + /annexin V − ; red) in Casp1 KO cells ( * P = 0.0265). ( K ) Flow cytometry analyzing CM from gemcitabine-treated WT or Casp1 knockout (Casp1 KO) T24 cells in the conversion between αSMA high/+ CAFs and αSMA − PDGFβ + iCAFs. ( L ) Corresponding IF costaining illustrating PDGFRβ (red) and αSMA (green) in CAFs exposed to CM-Gem from T24 WT and Casp1 KO cells. ( M ) Bar graph quantifying PDGFRβ + iCAFs, showing significant reduction after exposure to CM-Gem from Casp1 KO versus WT cells.

Journal: Science Advances

Article Title: Caspase-1–dependent pyroptosis converts αSMA + CAFs into collagen-III high iCAFs to fuel chemoresistant cancer stem cells

doi: 10.1126/sciadv.adt8697

Figure Lengend Snippet: ( A ) Schematic of CM from chemotherapy-treated cancer cells for subsequent treatment on CAFs in vitro. ( B ) Flow cytometry evaluating CM effects on αSMA high/+ CAFs and αSMA − PDGFRβ + iCAF conversion, in response to (i) base medium (2% FBS DMEM), (ii) CM from untreated T24 (CM-Veh), and (iii) CM from gemcitabine-treated T24 (CM-Gem). ( C and D ) Bar graphs quantifying αSMA high/+ CAFs (C) and αSMA − PDGFβ + iCAFs (D) upon treatment with CM-Veh and CM-Gem. ( E ) Flow cytometry histogram illustrating higher IL-6 expression in αSMA − PDGFRβ + iCAFs (blue) and αSMA + PDGFRβ + hybrid i/my CAFs (red) than other CAFs (green and yellow). ( F to H ) Western blot analysis of Casp1-dependent pyroptosis and apoptosis in gemcitabine-treated T24 cells by immunoblotting (IB): (F) Casp1 full-length (FL) protein, (G) Casp1 p20 and p10 cleavage (Cl) products indicating enzymatic activity, and (H) Caspase-3 (Casp3) FL, Cl-Casp3, and DNA repair protein poly(ADP-ribose) polymerase 1 (PARP1) FL and Cl PARP1 (Cl-PARP1), as apoptosis markers. ( I ) Flow cytometry of DAPI and annexin V costaining in WT and Casp1 knockout (Casp1 KO) T24 cells upon gemcitabine treatment. ( J ) Bar graph quantifying fractions in (I), showing reduced lytic cell death (DAPI + /annexin V − ; red) in Casp1 KO cells ( * P = 0.0265). ( K ) Flow cytometry analyzing CM from gemcitabine-treated WT or Casp1 knockout (Casp1 KO) T24 cells in the conversion between αSMA high/+ CAFs and αSMA − PDGFβ + iCAFs. ( L ) Corresponding IF costaining illustrating PDGFRβ (red) and αSMA (green) in CAFs exposed to CM-Gem from T24 WT and Casp1 KO cells. ( M ) Bar graph quantifying PDGFRβ + iCAFs, showing significant reduction after exposure to CM-Gem from Casp1 KO versus WT cells.

Article Snippet: After the last marker staining, the sections were incubated with DAPI in TBST (2 μg/ml) for 5 min, followed by three times of washing, and mounted with ProLong Gold Antifade Reagent with DAPI ( P36935 , Invitrogen).

Techniques: In Vitro, Flow Cytometry, Expressing, Western Blot, Activity Assay, Knock-Out