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. 2024 Feb 24;27(4):109288. doi: 10.1016/j.isci.2024.109288

Figure 3.

Figure 3

Depletion of LARP4A and LARP4B inhibits tumor formation in vivo

(A) Western blot analysis of endogenous LARP4A and LARP4B expression in PC3, MNNG/HOS, and MG63 cells 72h following transfection with two siRNAs each targeting LARP4A (siRNA1 and siRNA2, top panel) or LARP4B (siRNA1 and siRNA2, bottom panel) as indicated. Non-transfected (NTC) and non-targeting siRNA (siCon) controls are shown and GAPDH was used as a loading control.

(B) Representative images of xenograft tumors formed following subcutaneous injection of PC3 (top) or MNNG/HOS (bottom) cells following transfection with non-targeting siRNA (siControl) or siRNA-mediated knockdown of LARP4A (siLARP4A) or LARP4B (siLARP4B). Numbers below each panel denote the proportion of the engraftment of each cell line after 2 weeks. Scale bars, 1cm.

(C) Quantification of volumes of subcutaneous tumors shown in (B) formed by siLARP4A and siLARP4B knockdown PC3 (left) and MNNG/HOS (right) cells, 2 weeks post-engraftment and normalized to siControl (siCon) tumors. The data represent the mean ± SEM, n = 6–8 mice per condition. p values are indicated.

(D) Representative immunofluorescence images of pHH3 and DAPI staining in subcutaneous tumors formed two weeks following the engraftment of siControl, siLARP4A, or siLARP4B PC3 or MNNG/HOS cells. Scale bar, 100μm.

(E) Quantification of the proportion of cells within PC3 (left) and MNNG/HOS (right) xenografts expressing pHH3 shown in (D), as a percentage of the total number of cells (DAPI). Bars represent the mean ± SEM per tumor, n = 9–16 individual tumors from 6 to 8 mice. p values are indicated.

(F) Representative bioluminescence images of paratibial xenografts of siControl, siLARP4A, or siLARP4B MNNG/HOS cells the day after and 14 days post-engraftment.

(G) Quantification of the bioluminescence signal (top) and palpable tumor volume (bottom) in mouse tibiae over a 14 days time course. Data represent mean ± SEM, two tibiae measured from n = 8 mice per control or knockdown condition. p values are indicated (pink value indicates p value of the siControl vs. siLARP4A comparison; green of the siControl vs. siLARP4B comparison).

(H) Representative microCT scans of murine tibiae following engraftment with control (siControl), LARP4A (siLARP4A), or LARP4B (siLARP4B) knockdown MNNG/HOS cells. Arrows highlight areas of malignant bone deposits as a result of osteosarcoma tumor formation. Scale bars, 1mm.

(I) Representative histological images of tibiae 14 days post-engraftment with siControl, siLARP4A, or siLARP4B knockdown MNNG/HOS cells. Low magnification images show a clear reduction in tumor mass in both siLARP4A (I(d)) and siLARP4B (I(g)) knockdown xenografts compared to siControls (I(a)). High magnification images in panels I(b) and I(c) (siControl), I(e) and I(f) (siLARP4A) and I(h) and I(i) (siLARP4B) show areas of tumor tissue (T) that are markedly reduced in the LARP4A and 4B knockout xenografts. B, Bone, T, Tumor, BM, Bone Marrow. Asterisks indicate the cell injection sites. Arrows indicate the tumor perimeter which is reduced in the knockout xenografts. Scale bars, 500μm. See also Figure S3.