Note that "lsline superimposes a least-squares line on each scatter plot in the current axes".
That is, the second call to lsline returns two lines, since there are two scatter plots in the axes at that point in time.
To avoid creating redundant lines, make both scatter plots first, call lsline, and then set the properties of each line object returned:
all_subs_data = rand(10,150); % random data
scatter(all_subs_data(:,4),all_subs_data(:,144),"filled","o","MarkerFaceColor","r","SizeData",100, 'MarkerFaceAlpha',.1)
hold on
scatter(all_subs_data(:,5),all_subs_data(:,143),"filled","o","MarkerFaceColor","b","SizeData",100, 'MarkerFaceAlpha',.1)
h = lsline();
h(1).Color = 'r';
h(1).LineWidth = 3.0000;
h(2).Color = 'b';
h(2).LineWidth = 3.0000;
hold off
