Anti-SOX9 antibody [3C10] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SOX9 antibody [3C10] - BSA and Azide free (AB76997)

Immunoperoxidase analysis of SOX9 expression on formalin fixed, paraffin embedded human tonsil tissue using 0.7 μg/ml of ab76997.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-SOX9 antibody [3C10] - BSA and Azide free (AB76997)

Immunocytochemistry/Immunofluorescence analysis of SOX9 expression in HepG2 cells, using 10 μg/ml of ab76997.

• Flow Cyt

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Flow Cytometry - Anti-SOX9 antibody [3C10] - BSA and Azide free (AB76997)

Overlay histogram showing HepG2 cells stained with ab76997 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76997, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This image was generated using the ascites version of the product.

• WB

Unknown

Western blot - Anti-SOX9 antibody [3C10] - BSA and Azide free (AB76997)

This image was generated using the ascites version of the product.

All lanes:

Western blot - Anti-SOX9 antibody [3C10] - BSA and Azide free (ab76997) at 5 µg/mL

All lanes:

immunogen at 0.2 µg

Secondary

All lanes:

Goat Anti-Mouse IgG (H&L)-HRP Conjugate at 1/5000 dilution

Predicted band size: 56 kDa

Observed band size: 38 kDa

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• WB

CiteAb

Western blot - Anti-SOX9 antibody [3C10] - BSA and Azide free (AB76997)

Western Blotting using Anti-SOX9 antibody [3C10] - BSA and Azide free, ab76997. Publication image from López-Valero, I. et al., 2020, Theranostics, 32308772. Legend direct from paper.

Combination of MDK/ALK inhibitors with cannabinoids and TMZ strongly reduces the growth of GICs-derived xenografts. (A) Effect of daily oral administration of crizotinib (12.5 mg/kg, upper panel) or lorlatinib (25 mg/kg, bottom panel) on the volume of glioma xenografts generated by subcutaneous injection of 2 x 106 12O12-GICs (mean ± SEM on the last day of the treatment). **P < 0.01 from 12O12 vehicle-treated tumors. (B) Effect of daily oral administration of crizotinib (12.5 mg/kg) on the mRNA levels of NESTIN, MUSASHI-1 (MSI1), SOX2 and SOX9 (as determined by qPCR) of glioma xenografts generated by subcutaneous injection of 2 x 106 12O12-GICs (mean ± SEM). *P < 0.05 from 12O12 vehicle-treated tumors. (C) Effect of daily oral administration of lorlatinib (25 mg/kg) on the protein levels (as determined by Western blot) of NESTIN, MUSASHI-1 (MSI1), SOX2 and SOX9 in the tumor xenografts (V1-V6 : vehicle-treated animals; L1-L5 : lorlatinib-treated animals). (D) Effect of the treatment with crizotinib (0.5 µM) and TMZ (100 µM, upper panel or 20 µM bottom panel) on the self-renewal ability (as determined by LDA) of GH2 (upper panel) or 12O12 (bottom panel)-GICs (n=2). Full χ2 statistical analysis is included in LDA statistics section within the supplementary materials. (E) Effect of the treatment with THC : CBD (2.5 µM THC + 2.5 µM CBD) and MDK Ab. (40 µg/ml, left panel) or crizotinib (CZT, 0.25 µM, right panel) on the total number of cells (upper panels) and self-renewal capacity (bottom panels) of 12O12-GICs (n=3). **P < 0.01 and ***P < 0.001 from vehicle-treated cells; ###P < 0.001 from THC + CBD-treated cells; &P < 0.05 or &&P < 0.01 from MDK Ab.-treated cells or crizotinib-treated cells. Full χ2 statistical analysis is included in LDA statistics section within the supplementary materials. (F) Effect of the treatment with crizotinib (12.5 mg/kg daily oral administration) and TMZ (5 mg/kg twice a week IP administration) on the growth of glioma xenografts generated by subcutaneous injection of 2 x 106 12O12-GICs [mean ± SEM; n=5-6 mice for each condition]. Representative pictures of the tumor xenografts in the last day of the treatment are shown for each experimental condition. Symbols of significance are omitted for clarity except when the combined-treatment was significantly different from vehicle and each individual treatment (*P < 0.05 or **P <0.01 from each individual treatment). The rest of the statistical analysis can be found in Supplementary materials.

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• WB

CiteAb

Western blot - Anti-SOX9 antibody [3C10] - BSA and Azide free (AB76997)

Western Blotting using Anti-SOX9 antibody [3C10] - BSA and Azide free, ab76997. Publication image from López-Valero, I. et al., 2020, Theranostics, 32308772. Legend direct from paper.

Blockade of the MDK/ALK axis in GICs induces the autophagic degradation of SOX9. (A) LC3 and NESTIN immunostaining of GH2-GICs cells incubated with an anti-MDK antibody (MDK Ab., 40 µg/ml, 24 h) (n=3). Representative images (with a high magnification photomicrograph of the squared-pointed area of MDK Ab.-treated cells) are shown. Scale bar : 20 µm. (B) Quantification of the percentage of cells with LC3 dots within the population of NESTIN-positive or negative GH2-GICs (n=3). *P < 0.05 ***P < 0.001 from vehicle-treated cells; ##P < 0.01 from MDK Ab.-treated NESTIN-negative cells. ND : Non-detectable. (C) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 µg/ml) on MUSASHI1 (MSI1), CD133 and LC3-I/II protein levels of GH2-GICs stably transduced with a doxycycline-inducible shATG5 at different time points (n=3). A representative experiment is shown. (D) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 µg/ml) and the genetic inhibition of autophagy on the self-renewal ability (as determined by LDA) of GH2-GICs (n=2). #P < 0.05 from GH2 shATG5 (-Dox.) MDK Ab.-treated cells. Full χ2 statistical analysis is included in LDA statistics section within the supplementary materials. (E-F) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 µg/ml; 24 h, panel E) or TAE (0.75 µM, 24 h, panel F) on SOX9 and LC3-I/II protein levels of shC or shATG5-transduced GH2-GICs cultures untreated or pretreated with E64d (10 µM) and pepstatin A (PA, 10 µg/ml) for 1 h. A representative Western blot (upper panels) and the corresponding densitometric quantifications (bottom panels) are shown (n=5). **P < 0.01 from vehicle-treated cells; #P < 0.05 from MDK Ab.-treated shC cells.

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• WB

CiteAb

Western blot - Anti-SOX9 antibody [3C10] - BSA and Azide free (AB76997)

Western Blotting using Anti-SOX9 antibody [3C10] - BSA and Azide free, ab76997. Publication image from López-Valero, I. et al., 2020, Theranostics, 32308772. Legend direct from paper.

MDK/ALK signaling axis regulates the maintenance of the stem-like properties of GICs by controlling SOX9 protein levels. (A) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 µg/ml) or TAE (0.75 µM) for 24 h on SOX2, SOX4, SOX9 and CD133 protein levels of GH2-GICs. Left panel : a representative Western blot experiment is shown (n=5). Right panel : densitometric analysis of SOX9, SOX2 and SOX4 protein levels (mean fold change from vehicle ± SEM; n=5 for SOX9 and SOX2 and n=3 for SOX4). **P < 0.01 and ***P < 0.001 from vehicle-treated cells. NS : statistically non-significant differences. (B) Effect of SOX9 genetic inhibition [by nucleofection with a plasmid encoding a shcontrol (shC) or a SOX9-selective (shSOX9) shRNA] on the self-renewal ability (as determined by LDA) of GH2-GICs (n=3) **P < 0.01 from shC cells. (C) Effect of SOX9 genetic inhibition (72 h) on mRNA levels of a panel of stem cell associated genes (as determined by qPCR) of GH2-GICs. Data are expressed as mean fold change from shC cells (reference) ± SEM (n=3). *P < 0.05; **P < 0.01; **P < 0.001 from shC cells. (D) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 µg/ml) and nucleofection with a control plasmid (CP), or a plasmid encoding a murine SOX9 (SOX9) on the self-renewal ability (as determined by LDA) of GH2-GICs (n=2). #P < 0.05 from MDK Ab.-treated CP cells. Full χ2 statistical analysis is included in LDA statistics section within the supplementary materials. (E) Effect of the incubation with an anti-MDK antibody (MDK Ab., 40 µg/ml) on the mRNA levels of NESTIN, MUSASHI-1 (MSI1) and SOX9 (as determined by qPCR) of GH2-GICs nucleofected with a control plasmid (CP) or a plasmid encoding a murine SOX9 (SOX9) (72 h). *P < 0.05 from vehicle CP-treated cells.

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