FOXP1 promotes the proliferation and self-renewal of spermatogonial stem cells via the nuclear factor kappa B pathway

Figure 1 Forkhead box P1 was upregulated in obstructive azoospermia samples with normal spermatogenesis compared with non-obstructive azoospermia samples. A: Representative hematoxylin and eosin staining images of obstructive azoospermia and non-obstructive azoospermia testicular tissues; B: Immunohistochemical staining for the expression of forkhead box P1 in obstructive azoospermia and non-obstructive azoospermia testicular tissue samples. Scale bars = 100 μm, magnification 200 ×. OA: Obstructive azoospermia; NOA: Non-obstructive azoospermia.

Figure 2 Effect of forkhead box P1 inhibition on mice testicular morphology and weight. A: Hematoxylin and eosin staining of testicular tissues; B: Two testes were surgically excised and weighed 6 weeks after the mice were infected with sh-FOXP1 lentivirus. Scale bars = 100 μm, magnification 200 ×. n = 6, ^cP < 0.001. NC: Negative control; FOXP1: Forkhead box P1.

Figure 3 Forkhead box P1 knockdown inhibited the self-renewal of spermatogonial stem cells in mice. A and B: Immunohistochemical staining showing the protein level changes of ETS variant gene 5 and promyelocytic leukemia zinc finger in mice testes after transfection with sh-FOXP1 lentivirus; C and D: Immunofluorescence staining of ETS variant gene 5 and promyelocytic leukemia zinc finger in mice testes after transfection with sh-FOXP1 lentivirus. Scale bars = 100 μm. FOXP1: Forkhead box P1; NC: Negative control; ETV5: ETS variant gene 5; PLZF: Promyelocytic leukemia zinc finger; DAPI: 4’6-diaminophenylindole.

Figure 4 Forkhead box P1 promoted the proliferation and self-renewal of spermatogonial stem cells via the nuclear factor kappa B pathway. A: Cell Counting Kit-8 assay showing the proliferation of spermatogonial stem cells (SSCs) in the control and sh-FOXP1 groups; B: Cell Counting Kit-8 assay showing the proliferation of SSCs in the control, nuclear factor kappa B (NF-κB) inhibitor (SC75741), FOXP1 overexpression (FOXP1-OE), and FOXP1-OE + NF-κB inhibitor groups; C: Western blot analysis showing the expression of proliferation and self-renewal proteins proliferating cell nuclear antigen, ETS variant gene 5, and promyelocytic leukemia zinc finger in the control and sh-FOXP1 groups; D: Western blot analysis showing the expression of proliferation and self-renewal proteins proliferating cell nuclear antigen, ETS variant gene 5, and promyelocytic leukemia zinc finger in the control, NF-κB inhibitor, FOXP1-OE, and FOXP1-OE + NF-κB inhibitor groups; E: Western blot analysis showing the expression of NF-κB p65 and phosphorylated-NF-κB p65 in the control and sh-FOXP1 groups; F: Western blot analysis showing the expression of NF-κB p65 and phosphorylated-NF-κB p65 in the control, NF-κB inhibitor, FOXP1-OE, and FOXP1-OE + NF-κB inhibitor groups. n ≥ 3, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. NS: Not significant; FOXP1: Forkhead box P1; NC: Negative control; ETV5: ETS variant gene 5; PLZF: Promyelocytic leukemia zinc finger; NF-κB: Nuclear factor kappa B; p-NF-κB p65: Phosphorylated nuclear factor kappa B p65; PCNA: Proliferating cell nuclear antigen.